Modified nucleoside phosphoramidites

ABSTRACT

The present disclosure relates to compounds and compositions containing 5′-phosphoramidite nucleoside monomers of formulae (I) and (II), and methods of making and use, wherein the substituents are as defined in the appended claims.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a U.S. application claiming the benefit ofpriority to U.S. Provisional Application No. 62/558,763, filed Sep. 14,2017, the entirety of which is hereby incorporated by reference.

BACKGROUND

Oligonucleotide synthesis is extremely important for providing access tocustom-made oligonucleotides of the desired sequence. To obtain thedesired oligonucleotide, the building blocks (monomers) are sequentiallycoupled to the growing oligonucleotide chain in the order required bythe sequence of the product.

Modified oligonucleotides, having modifications at, e.g., the 2′ and/or3′ position have received increased interest over the past years asuseful in, e.g., therapeutic applications. While synthetic methods forconstructing modified oligonucleotides exist, there is a need foradditional synthetic options to synthesize a diverse array of modifiedoligonucleotides. New monomeric nucleosides of high purity, and whichare capable of being synthesized at higher yield and at high volume, areneeded to meet the demand for new modified oligonucleotides.

Monomers described herein meet this need by providing novel monomersuseful for the synthesis of modified oligonucleotides.

SUMMARY

The present disclosure relates to compounds and compositions containing5′-phosphoramidite nucleoside monomers, and methods of making and use.

The present disclosure is directed to compounds having a structurerepresented by Formula (I):

X is O or NH; Y is selected from the group consisting of O-PG, —O(CR⁴₂)_(a)CR⁴ ₃, —O(CR⁴ ₂)_(b)OCR⁴ ₃ and —O(CR⁴ ₂)_(b)—CR⁴═CR⁴ ₂; Z is H; PGis a protecting group; B is a natural or an unmodified nucleobase or amodified nucleobase or a protected version thereof; each R¹ isindependently C₁₋₆ alkyl or cycloalkyl; R² is CH₂CH₂CN or C₁₋₆ alkyl; orone R¹ and R² together form an optionally substituted C₁₋₆ cycloalkyl;R³ is H or PG; R⁴ is independently in each instance H or F; a is aninteger of 0-2; and b is an integer of 1-3. In some embodiments, when Xis O, then Y is —O(CR⁴ ₂)_(a)CR⁴ ₃, —O(CR⁴ ₂)_(b)OCR⁴ ₃ or O(CR⁴₂)_(b)—CR⁴═CR⁴ ₂.

The present disclosure is also directed to compounds having a structurerepresented by Formula (II):

X is O or NH; Z is H; B is a natural or an unmodified nucleobase or amodified nucleobase or a protected version thereof; R¹ is independentlya C₁₋₆ alkyl or cycloalkyl; R² is CH₂CH₂CN or a C₁₋₆ alkyl; or one R¹and R² together form an optionally substituted C₁₋₆ cycloalkyl; R³ is Hor a protecting group; A is —(CR′R′)₁₋₂—; and R¹ is independently ineach instance H or Me.

In some embodiments, X is NH, Y is O-PG, OEt or O-methoxyethoxy and Z isH.

In some embodiments, R¹ is C₂₋₃ alkyl. In some embodiments, R¹ isisopropyl.

In embodiments, R³ is PG. In some embodiments, PG is trityl. In someembodiments, PG is monomethoxytrityl (MMTr) or trilyl when X is NH. Insome embodiments, PG is 4,4′-dimethoxytrityl (DMTr) or trityl when X isO.

In some embodiments, B is selected from adenine (A), guanine (G),thymine (T), cytosine (C), uracil (U) and 5-methylcytosine (5-me-C), ora protected version thereof. In some embodiments, B is selected from6-N-benzoyladenosine (A^(Bz)), 4-N-benzoylcytidine (C^(Bz)), and2-N-isobutyrylguanosine (G^(iBu)).

The present disclosure is also directed to a composition comprising atleast one compound of Formula (I) or Formula (II). In embodiments, thecomposition further comprises one or more other agents selected from thegroup consisting of reactants, solvents and impurities. In embodiments,the composition comprises less than 5%, less than 4%, less than 3%, lessthan 2% or less than 1% impurities. In some embodiments, the compositionhas a purity of >95%, e.g., 96, 97, 98, 99%, or more.

The present disclosure is further directed to methods of making anoligonucleotide comprising at least one modified nucleoside. The methodcomprises reacting a compound of Formula (I) with another monomer toform the oligonucleotide.

DETAILED DESCRIPTION

The present disclosure is described herein in greater detail.

Compounds of the Present Disclosure

Compounds of the present disclosure include one or more compounds of thefollowing Formula (I) and/or (II):

whereinX is O or NH; Y is selected from the group consisting of O-PG, —O(CR⁴₂)_(a)CR⁴ ₃, —O(CR⁴ ₂)_(b)OCR⁴ ₃ (e.g., OEt, O-methoxyethoxy, and OCF₃);Z is H; PG is a protecting group; B is a natural or an unmodifiednucleobase or a modified nucleobase or a protected version thereof; eachR¹ is independently C₁₋₆ alkyl or cycloalkyl; R² is CH₂CH₂CN or C₁₋₆alkyl; or one R¹ and R² together form an optionally substituted C₁₋₆cycloalkyl; R³ is H or PG; R⁴ is independently in each instance H or F;a is an integer of 0-2 (e.g., 0, 1 or 2); and b is an integer of 1-3(e.g., 1, 2 or 3), A is —(CR′R′)₁₋₂—; and R′ is independently in eachinstance H or Me.

In some embodiments, when X is O, then Y is —O(CR⁴ ₂)_(a)CR⁴ ₃, —O(CR⁴₂)_(b)OCR⁴ ₃ or —O(CR⁴ ₂)_(b)—CR⁴═CR⁴ ₂. In some embodiments, X is O, Yis OEt or O-methoxyethoxy and Z is H. In other embodiments, X is NH, Yis OP, OEt or O-methoxyethoxy and Z is H.

In nucleotides of Formula (I), Y is —O(CR⁴ ₂)₂₀CR⁴ ₃. In someembodiments, R⁴ is H in each instance. In other embodiments, at leastone R⁴ is F, for example, 1, 2, 3, 4, 5, 6, or 7 R⁴s are F. In someembodiments, CR⁴ ₃ contains 1, 2 or 3 F atoms. For example, inembodiments, Y is selected from the group consisting of —OCH₂CH₂OCH₃ (orMOE), —OCF₂CH₂OCH₃, —OCH₂CF₂OCH₃, —OCH₂CH₂OCF₃, —OCF₂CF₂OCH₃,—OCH₂CF₂OCF₃, —OCF₂CH₂OCF₃, —OCF₂CF₂OCF₃, —OCHFCH₂OCH₃, —OCHFCHFOCH₃,—OCHFCH₂OCFH₂, —OCHFCH₂OCHF₂ and —OCH₂CHFOCH₃.

In the nucleotide of Formula (I), Y is —OCR⁴ ₃, —O(CR⁴ ₂)₁₋₃OCR⁴ ₃, or—O(CR⁴ ₂)₁₋₂CR⁴ ₃. In some embodiments, Y is —OCR⁴ ₃ or —OCR⁴ ₂CR⁴ ₃. Insome embodiments, R⁴ is H in each instance. In other embodiments, atleast one R⁴ is F, for example, 1, 2, 3, 4, or 5 R⁴s are F. In someembodiments, OCR⁴ ₃ contains 1, 2 or 3 F atoms. For example, inembodiments, Y is selected from the group consisting of —OCH₃ (or Me),—OCFH₂, —OCHF₂, OCF₃, —OCH₂OCH₃, —OCFH₂OCH₃, —OCHF₂₀CH₃, —OCF₃OCH₃,—OCH₂OCFH₂, —OCH₂OCHF₂, —OCH₂OCF₃, —OCFH₂OCH₃, —OCFH₂OCFH₂, —OCFH₂OCHF₂,—OCFH₂OCF₃, —OCHF₂OCH₃, —OCHF₂OCFH₂, —OCHF₂OCHF₂, —OCHF₂OCF₃, —O(C R⁴₂)₃₀C R⁴ ₃, —OCH₂CH₃ (or Et), —OCFH₂CH₃, —OCHF₂CH₃, —OCF₃CH₃, —OCH₂CFH₂,—OCH₂CHF₂, —OCH₂CF₃, —OCFH₂CH₃, —OCFH₂CFH₂, —OCFH₂CHF₂, —OCFH₂CF₃,—OCHF₂CH₃, —OCHF₂CFH₂, —OCHF₂CHF₂, —OCHF₂CF₃, —OCH₂CH₂CH₃, OCF₂CH₂CH₃,OCH₂CF₂CH₃, OCH₂CH₂CF₃, OCF₂CF₂CH₃, OCH₂CF₂CF₃, OCF₂CH₂CF₃, OCF₂CF₂CF₃,OCHFCH₂CH₃, OCHFCHFOCH₃, OCHFCH₂CFH₂, OCHFCH₂CHF₂ and OCH₂CHFCH₃. Inembodiments, Y is —OCH₃ (or Me) or —OCH₂CH₃ (or Et).

In some embodiments of Formula (I), Y is —O(CR⁴ ₂)_(b)—CR⁴═CR⁴ ₂. Insome embodiments, b is 1, 2 or 3. In some embodiments, R⁴ is H in eachinstance. In other embodiments, at least one R⁴ is F, for example, 1, 2,3, 4, 5, 6, or 7 R⁴s are F. In some embodiments, a CR⁴ ₂ contains 1 or 2F atoms, e.g. an internal R⁴ ₂ contains 1 or 2 F atoms, or the terminalR⁴ ₂ contains 1 or 2 F atoms.

In some embodiments, R¹ is a C₂₋₃ alkyl (e.g., an ethyl or isopropyl).

In some embodiments, R³ is a protecting group, PG. Protecting groups mayinclude an amine or alcohol protecting group, such as a silyl protectinggroup (e.g., tert-Butyldimethylsilyl ether (TBMDS),tert-Butyldiphenylsilyl (TBDPS), Triisopropylsilyl ether (TIPS)) ormonomethoxytrityl (MMTr) or 4,4′-dimethoxytrityl (DMTr) or tritolyl orany other suitable protecting groups such as those in Wuts, Peter GM,and Theodora W. Greene. Greene's protective groups in organic synthesis.John Wiley & Sons, 2006. In some embodiments, when more than one alcoholand/or amine is protected, the alcohols and/or amines may beorthogonally protected.

In compounds of Formula (II), A is —(CR′R′)₁₋₂—. In some embodiments, Ais —(CR′R′)— in other embodiments, A is —(CR′R′)₂—. R′ is independentlyin each instance H or Me. In some embodiments, one R′ is Me and theremaining R′ are H. In other embodiments all R′ are H. In someembodiments, X is NH in Formula (II). In some embodiments, A is not—(CR′R′)₁— when R′═H and X═NH.

The natural or unmodified nucleobase or a modified nucleobase or aprotected version thereof is not particularly limited. In someembodiments, B is selected from purine bases, such as, adenine (A) andguanine (G), diaminopurine (DAP) and pyrimidine bases, such as, thymine(T), cytosine (C) and uracil (U) and/or other synthetic and naturalnucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine.In some embodiments, B is a protected nucleobase, such as a protected U,T, C, 5meC, A or G. Protection moieties are known in the art, and arenot particularly limited, but include, for example, amino protectinggroups (e.g., acetamide protecting groups, such as benzamide andisobutyramide). In some embodiments, B is 6-N-benzoyladenosine (A^(Bz)),4-N-Benzoylcytidine (C^(Bz)), 2-N-isobutyrylguanosine (G^(iBu)).

In some embodiments, the compound is selected from the following table.

X Y Z B R¹ R² R³ NH OP H U/T/C/5meC/A/G/DAP iPr CH₂CH₂CN MMTr NH OEt HU/T/C/5meC/A/G/DAP iPr CH₂CH₂CN MMTr NH O- H U/T/C/5meC/A/G/DAP iPrCH₂CH₂CN MMTr methoxy ethyl NH OCF₃ H U/T/C/5meC/A/G/DAP iPr CH₂CH₂CNMMTr O OEt H U/T/C/5meC/A/G/DAP iPr CH₂CH₂CN DMTr O O- HU/T/C/5meC/A/G/DAP iPr CH₂CH₂CN DMTr methoxy ethyl *B may be a protectednucleobase (e.g., A^(Bz), C^(Bz), G^(iBu)).

In some embodiments, the compound is selected from the following table.

Compositions

The present disclosure also encompasses compositions comprising acompound of the present disclosure and one or more reactants or solventsor impurities.

In some embodiments, the compositions comprising a compound of thepresent disclosure have a purity of 95%, 96%, 97%, 98%, 99%, 99.5%,99.6%, 99.7%, 99.8%, 99.9%, 99.95% or more.

Methods of Use

The compositions of the present disclosure may be used as syntheticprecursors for oligonucleotides comprising modified nucleosides, e.g.,those in U.S. Provisional Applications 62/420,801, 62/394,737,62/394,738 and 62/394,739, each of which is incorporated by reference.

The modified oligonucleotides may be synthesized by methods in the art,e.g., on an ABI-394 synthesizer using the 93-step cycle written withmodifications to deblock, coupling and wait steps. The solid support canbe 3′-NHTr-5′-LCAA-CPG. Each oligonucleotide is individually synthesizedusing methods described herein.

Methods of Making

The compositions of the present disclosure may be synthesized bysynthetic procedures such as those set forth in the below Examples.

Some embodiments include 2′-O-alkylation of a starting material, such asthe following:

where PG is a protecting group, such as trityl and BP is a protectednucleobase, protected with, e.g., PMB. The compound is alkylated,followed by deprotection of the PGO and reprotection with, e.g., a Bzmoiety, followed by deprotection of the nucleobase and subsequentconversion of the N₃ to N—R₃.

Some embodiments include protection of a compound, such as that in theprevious paragraph where the nucleobase is a purine, with C-6-OBn. Insome embodiments, this reduces de-purination during deprotection of,e.g., 5′-O-DMTr and C-2-NH-MMTr group under acidic conditions. Otherembodiments include protection of the exocyclic amine group in acompound, such as that in the previous paragraph where the nucleobase isa purine, with a bulky protecting group such as 4-monomethoxy tritylgroup to achieve 2′-O-alkylation in high yield.

Some embodiments include protection of a compound, such as that in theprevious paragraph where the nucleobase is a pyrimidine to achieve2′-O-alkylation by protecting the pyrimidine with a PMB moiety.

Definitions

It is to be understood that the terminology used herein is for thepurpose of describing particular embodiments only and is not intended tolimit the scope of the present invention. The following definitionsshall apply unless otherwise indicated.

“Pharmaceutically acceptable” refers to a material that is notbiologically or otherwise undesirable, i.e., the material may beincorporated into a pharmaceutical composition administered to a patientwithout causing any undesirable biological effects or interacting in adeleterious manner with any of the other components of the compositionin which it is contained. When the term “pharmaceutically acceptable” isused to refer to a pharmaceutical carrier or excipient, it is impliedthat the carrier or excipient has met the required standards oftoxicological and manufacturing testing or that it is included on theInactive Ingredient Guide prepared by the U.S. and Drug administration.

“Modified nucleoside” refers to a nucleoside having, independently, amodified sugar moiety and/or modified nucleobase.

“Unmodified” or “natural” nucleobases include the purine bases adenine(A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C)and uracil (U). “Modified nucleobases” include other synthetic andnatural nucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and otheralkyl derivatives of adenine and guanine, 2-propyl and other alkylderivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and2-thiocytosine, 5-halouracil and cytosine, 5-propynyl (—C≡C—CH₃) uraciland cytosine and other alkynyl derivatives of pyrimidine bases, 6-azouracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil,8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other8-substituted adenines and guanines, 5-halo particularly 5-bromo,5-trifluoromethyl and other 5-substituted uracils and cytosines,7-methylguanine and 7-methyladenine, 2-F-adenine, 2-amino-adenine,8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine and3-deazaguanine and 3-deazaadenine. Further modified nucleobases includetricyclic pyrimidines such as phenoxazinecytidine(1H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), phenothiazinecytidine (1H-pyrimido[5,4-b][1,4]benzothiazin-2(3H)-one), G-clamps suchas a substituted phenoxazine cytidine (e.g.9-(2-am-oelhoxy)-H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), carbazolecytidine (2H-pyrimido[4,5-b]indol-2-one), pyridoindole cytidine(H-pyrido[3,2,5]pyrrolo[2,3-d]pyrimidin-2-one). Modified nucleobases mayalso include those in which the purine or pyrimidine base is replacedwith other heterocycles, for example 7-deaza-adenine, 7-deazaguanosine,2-aminopyridine and 2-pyridone.

In some embodiments, the modified nucleobase is selected from the groupconsisting of 5-methylcytosine, 2,6-diaminopurine, 5-methyluracil, and ag-clamp. In some embodiments, the g-clamp is

It is noted that, as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural referents unless thecontext clearly dictates otherwise. It is further noted that the claimsmay be drafted to exclude any optional element. As such, this statementis intended to serve as antecedent basis for use of such exclusiveterminology as “solely”, “only” and the like in connection with therecitation of claim elements, or use of a “negative” limitation.

The term “about” will be understood by persons of ordinary skill in theart and will vary to some extent depending upon the context in which itis used. If there are uses of the term which are not clear to persons ofordinary skill in the art given the context in which it is used, “about”will mean up to plus or minus 10% of the particular term. Certain rangesare presented herein with numerical values being preceded by the term“about”. The term “about” is used herein to provide literal support forthe exact number that it precedes, as well as a number that is near toor approximately the number that the term precedes. In determiningwhether a number is near to or approximately a specifically recitednumber, the near or approximating unrecited number may be a number,which, in the context in which it is presented, provides the substantialequivalent of the specifically recited number.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range, is encompassed within the invention. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges and are also encompassed within the invention, subject toany specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either orboth of those included limits are also included in the invention.

This disclosure is not limited to particular embodiments described, assuch may, of course, vary. It is also to be understood that theterminology used herein is for the purpose of describing particularembodiments only, and is not intended to be limiting, since the scope ofthe present invention will be limited only by the appended claims.

As will be apparent to those of skill in the art upon reading thisdisclosure, each of the individual embodiments described and illustratedherein has discrete components and features which may be readilyseparated from or combined with the features of any of the other severalembodiments without departing from the scope or spirit of the presentinvention. Any recited method can be carried out in the order of eventsrecited or in any other order that is logically possible.

All publications and patents cited in this specification are hereinincorporated by reference as if each individual publication or patentwere specifically and individually indicated to be incorporated byreference and are incorporated herein by reference to disclose anddescribe the methods and/or materials in connection with which thepublications are cited. The citation of any publication is for itsdisclosure prior to the filing date and should not be construed as anadmission that the present invention is not entitled to antedate suchpublication by virtue of prior invention. Further, the dates ofpublication provided may be different from the actual publication datesthat may need to be independently confirmed.

EXAMPLES

The following examples illustrate certain embodiments of the presentdisclosure to aid the skilled person in practicing the disclosure.Accordingly, the examples are in no way considered to limit the scope ofthe disclosure.

Examples 1-4

The appropriately protected 2′-O-methoxyethyl-3′-aminonucleoside-5′-phosphoramidite building blocks (examples 1,2, 3, and 4 were prepared after chemical transformations shown inSchemes 1, 2, 3, 4, and 5.

Scheme 1 discloses synthesis of uracil-based3′-NH-MMTr-2′-O-methoxyethyl phosphoramidites (such as example 1). Thekey 3′-azido-2′-methoxyethyl Intermediate 3-3 was obtained in low yieldsvia anhydrous Intermediate 3-2.

Due to low yielding alkylation, 3-1 was reacted with BOMCl/DBU to giveN-3 protected Intermediate 3-4, which was alkylated by using2-bromoethyl methyl ether/Ag₂O/NaI/DMF to give 2′-O-methoxyethylderivative 3-5. Deprotection of N-3-BOM group using hydrogenationcondition (Pd/C/H₂) resulted in 10-20% desired 3′-amino Intermediate3-6a along with significant over reduced side product 3-6b.

Scheme 2 discloses an alternative approach to achieve 2′-O-alkylation inhigh yield. Intermediate 3-1 was treated with PMBCl/DBU/DMF to give N-3protected Intermediate 4-2, which was subjected to 2′-O alkylation using2-bromoethyl methyl ether/Ag₂O/NaI/DMF to provide a 2′-O-methoxyethylderivative (Intermediate 4-3). 5′-de-tritylation of Intermediate 4-3 andre-protection of its 5′-hydroxyl group using benzoyl chloride providedIntermediate 4-5

De-protection of PMB group of Intermediate 4-5 in mild conditions togive Intermediate 4-6. 3′-azido group of intermediate 4-6 was reduced toan amine, which was then immediately protected, such as reaction with4-monomethoxytritylchloride, to give 4-8. The 5′-benzyl ester was thencleaved using an alkaline solution, followed by phosphitylation usingknown methods to give the desired 2′-O-methoxyethoxy uridinephosphoramidite monomer 4-10.

Preparation of Intermediate (4-2):

To a solution of 3-1 (45.30 g, 88.56 mmol) in DMF (120.00 mL) was addedPMBCl (20.80 g, 132.84 mmol) and DBU (44.61 g, 177.12 mmol), the mixturewas stirred at r.t. for 2 h. Water was added, extracted with EA. Theorganic layer was concentrated and purified by column to give 4-2 (52.00g, 82.32 mmol) as a white solid. ESI-LCMS: m/z 632.3 [M+H]⁺.

Preparation of Intermediate (4-3):

To a solution of 4-2 (50.00 g, 79.15 mmol) in DMF (120.00 mL) was added2-Bromoethyl methyl ether (16.50 g, 118.73 mmol) and Ag₂O (18.34 g,79.15 mmol, 2.57 mL), then NaI (5.93 g, 39.58 mmol) was added. Thereaction mixture was stirred at r.t. for 12 h. LC-MS showed work well.Filtered and added water and EA, the organic layer was concentrated andpurified by column to give 4-3 (52.00 g, 75.39 mmol) as a colorless oil.ESI-LCMS: m/z 690.4 [M+H]⁺.

Preparation of Intermediate (4-4):

To a solution of 4-3 (52.00 g, 75.39 mmol) in DCM (200.00 mL) was addedTFA (150.00 mL). The mixture was stirred at r.t. for 1 h. The reactionmixture was slowly added to cold NH₄OH, extracted with DCM. The organiclayer was concentrated and purified to give 4-4 (31.00 g, 69.28 mmol) asa colorless oil. ESI-LCMS: m/z 448.2 [M+H]⁺. ¹H-NMR (DMSO-d₆, 400 MHz):δ ppm 8.02 (d, J=8.12 Hz, 1H), 7.26-7.23 (m, 2H), 6.87-6.84 (m, 2H),5.87-5.81 (m, 2H), 5.38 (t, J=5.0 Hz, 1H), 4.96-4.85 (m, 2H), 4.36-4.34(m, 1H), 4.17-4.14 (m, 1H), 4.00-3.97 (m, 1H), 3.83-3.77 (m, 1H),3.75-3.72 (m, 1H), 3.71 (s, 3H), 3.70-3.68 (m, 1H), 3.61-3.56 (m, 1H),3.45-3.43 (m, 2H), 3.18 (s, 3H).

Preparation of Intermediate (4-5):

To a solution of 4-4 (31.00 g, 69.28 mmol) in Pyridine (200.00 mL) wasadded BzCl (13.14 g, 93.87 mmol), the reaction mixture was stirred atr.t. for 15 min and concentrated and purified by column to give 4-5(35.10 g, 63.8 mmol) as a white solid. ESI-LCMS: m/z 552.2 [M+H]⁺.

Preparation of Intermediate (4-6):

To a solution of 4-5 (35.10 g, 63.8 mmol) in acetonitrile (300.00 mL)and water (100.00 mL) was added Ceric ammonium nitrate (105 g, 191.40mmol), the reaction mixture was stirred at r.t. for 12 h andconcentrated and extracted with EA. The organic layer was concentratedand purified by column to give 4-6 (27.5 g, 63.75 mmol) as a yellowsolid. ESI-LCMS: m/z 432.2 [M+H]⁺.

Preparation of Intermediate (4-7):

To a solution of 4-6 (27.50 g, 63.75 mmol) in THF (500.00 mL) was addedPd/C (3.00 g), the reaction mixture was stirred at r.t. for 12 h andfiltered and concentrated to give 4-7 (25.00 g, 61.67 mmol) as a yellowsolid. ESI-LCMS: m/z 406.2 [M+H]⁺.

Preparation of Intermediate (4-8):

To a solution of 4-7 (25.00 g, 61.67 mmol) in DCM (300.00 mL) was addedMMTrCl (28.49 g, 92.51 mmol) and Collidine (14.95 g, 123.34 mmol), thenAgNO₃ (15.7 g, 92.5 mmol) was added. The reaction mixture was stirred atr.t. for 1 h., and filtered and the organic layer was washed water,dried over Na₂SO₄ and purified by silica gel column to give 4-8 (33.00g, 48.69 mmol) as a yellow solid.

Preparation of Intermediate (4-9):

To a solution of 4-8 (14.50 g, 21.39 mmol) was added 1 N NaOH inmethanol (200 mL) in water (20 mL), the reaction mixture was stirred atr.t. for 1 h. and concentrated and extracted with DCM, the organic layerwas concentrated and purified by silica gel column to give 4-9 (11.50 g,20.05 mmol) as a white solid. 1H-NMR (DMSO-d₆, 400 MHz): δ ppm 11.26 (s,1H), 7.95 (d, J=8.4 Hz, 1H), 7.47-7.44 (m, 4H), 7.34-7.17 (m, 8H), 6.82(d, J=8.8 Hz, 2H), 5.50-5.48 (m, 2H), 5.13 (t, J=3.6 Hz, 1H), 4.05-3.98(m, 3H), 3.78 (s, 3H), 3.52-3.49 (m, 1H), 3.34-3.32 (m, 2H), 3.14 (s,3H), 3.08-3.04 (m, 1H), 2.89-2.86 (m, 1H), 2.70 (d, J=10.0 Hz, 1H), 1.51(d, J=4.4 Hz, 1H).

Preparation of (4-10):

To a solution of 4-9 (11.50 g, 20.05 mmol) in DCM (100.00 mL) was addedDMAP (489.85 mg, 4.01 mmol) and DIPEA (10.36 g, 80.19 mmol, 14.01 mL).Then CEPCl (5.70 g, 24.06 mmol) was added to the solution. The mixturewas stirred at r.t. for 30 min. The reaction was quenched with saturatedNaHCO₃. The organic layer was washed with brine, dried over Na₂SO₄,concentrated to give the crude product. The crude product was purifiedby Flash-Prep-HPLC. The product was dissolved in anhydrous toluene andconcentrated for three times. Then the product was dissolved anhydrousacetonitrile and concentrated for three times. This resulted in 13 g togive 4-10 as a white solid. MS m/z [M−H]⁻ (ESI): 772.3; ¹H-NMR (CDCl₃,400 MHz): 9.01 (s, 1H), 8.07-7.61 (m, 1H), 7.53-7.41 (m, 6H), 7.29-7.15(m, 5H), 6.79-6.76 (m, 2H), 5.63-5.57 (m, 2H), 4.27-4.15 (m, 2H),4.06-3.95 (m, 1H), 3.85-3.77 (m, 1H), 3.75 (s, 3H), 3.69-3.35 (m, 7H),3.23 (d, J=4 Hz, 1H), 2.26-2.91 (m, 3H), 2.59 (t, J=6.4 Hz, 1H),1.75-1.39 (m, 1H), 1.21-1.11 (m, 12H). ³¹PNMR (162 MHz, CDCl₃): 149.10,148.26.

Example 2

The 2′-O-methoxyethoxy-NH-benzoyl-cytosine phosphoramidite compound 5-4was obtained by conversion of uridine intermediate 4-8 into 3′-aminocytidine analogue 5-1 followed by phosphitylation using known protocolsto give the desired 2′-O-methoxyethoxy cytidine phosphoramidite monomer5-4 as shown below in scheme 3.

Preparation of Intermediate (5-1):

To a solution of 4-8 (18.50 g, 27.30 mmol) in acetonitrile (250.00 mL)was added TPSCl (16.49 g, 54.60 mmol) and DMAP (6.67 g, 54.60 mmol),then TEA (5.52 g, 54.60 mmol, 7.56 mL) was added to the solution. Thereaction mixture was stirred at r.t. for 5 h under N₂. NH₄OH (50.00 mL)was added to the reaction mixture. The mixture was stirred at r.t. for12 h. The solution was concentrated and extracted with EA. The organiclayer was washed by brine and dried over Na₂SO₄. The organic layer wasconcentrated and purified by silica gel column to give 5-1 (16.00 g,23.64 mmol) as a yellow solid.

Preparation of Intermediate (5-2):

To a solution of 5-1 (16.00 g, 23.64 mmol) in Pyridine (100.00 mL) wasadded BzCl (4.96 g, 35.46 mmol) at 0° C. The mixture was stirred at r.t.for 1 h. The solution was concentrated and purified by silica gel columnto give 5-2 (17.40 g, 22.28 mmol) as a white solid.

Preparation of Intermediate (5-3):

Compound 5-2 (17.40 g, 22.28 mmol) was added to 180 mL of 1 N NaOHsolution in Pyridine/MeOH/H₂O (65/30/5) at 0° C. The suspension wasstirred at 0° C. for 15 min. The reaction mixture was quenched byaddition of sat. NH₄Cl solution. The solution was extracted with EA andthe combined organic layers were washed with sat. NaHCO₃ solution,brine, dried over Na₂SO₄, filtered and concentrated. The residue waspurified by column to give 5-3 (12.50 g, 18.47 mmol) as white solid.1H-NMR (DMSO-d₆, 400 MHz): δ ppm 12.25 (s, 1H), 8.53 (d, J=7.6 Hz, 1H),8.01 (d, J=5.2 Hz, 2H), 7.64-7.60 (m, 1H), 7.52-7.42 (m, 6H), 7.31 (d,J=8.8 Hz, 2H), 7.26-7.14 (m, 7H), 6.79 (d, J=8.8 Hz, 2H), 5.55 (s, 1H),5.23 (t, J=3.6 Hz, 1H), 4.09-3.97 (m, 3H), 3.73 (s, 3H), 3.70-3.66 (m,1H), 3.38-3.34 (m, 2H), 3.17 (s, 3H), 3.11-3.05 (m, 1H), 2.96-2.91 (m,1H), 2.68 (d, J=10.8 Hz, 1H), 1.49 (d, J=4 Hz, 1H).

Preparation of (5-4):

To a solution of 5-3 (12.50 g, 18.47 mmol) in DCM (100.00 mL) was addedDMAP (451.30 mg, 3.69 mmol) and DIPEA (9.55 g, 73.88 mmol, 12.90 mL),then CEPCl (5.25 g, 22.16 mmol) was added. The mixture was stirred atr.t. for 30 min. The reaction was quenched with saturated NaHCO₃. Theorganic layer was washed with brine, dried over Na₂SO₄, concentrated togive the crude product. The crude was by Flash-Prep-HPLC. The productwas dissolved in anhydrous toluene and concentrated for three times.Then the product was dissolved anhydrous acetonitrile and concentratedfor three times. This resulted in 13 g to give 5-4 as a white solid. MSm/z [M−H]⁻ (ESI): 875.4. ¹H-NMR (400 MHz, CDCl₃): δ ppm 8.64-8.20 (m,2H), 7.90-7.88 (m, 2H), 7.62-7.58 (m, 1H), 7.53-7.39 (m, 8H), 7.25-7.15(m, 6H), 6.78-6.74 (m, 2H), 5.69 (d, J=1.72 Hz, 1H), 4.37-4.21 (m, 2H),4.10-4.03 (m, 1H), 3.90-3.79 (m, 2H), 3.75 (d, J=1.64 Hz, 3H), 3.68-3.52(m, 3H), 3.46-3.42 (m, 2H), 3.26 (d, J=1.2 Hz, 3H), 3.17-2.97 (m, 2H),2.94-2.87 (m, 1H), 2.67-2.48 (m, 2H), 1.79-1.51 (m, 1H), 1.26-1.18 (m,12H). ³¹PNMR (162 MHz, CDCl₃): 148.93, 148.03

Example 3

The synthesis of the 2′-O-methoxyethyl adenosine analogue 6-10 wasachieved as shown below in scheme 4. The intermediate 6-2 under basiccondition (NH₃/MeOH) resulted in diol 6-3, which then upon protection of5′-hydroxy group using TBDPSCl to give 6-4. Intermediate 6-4. Then, 2′-Oalkylation of 6-4 using 2-bromoethyl methyl ether/NaH/DMF to give2′-O-methoxyethyl derivative 6-5 without the protection of C-6-exocyclicamine of 6-4. In an inventive way selective alkylation of 2′—OH group ofintermediate 6-4 was achieved.

3′-Azido group of intermediate 6-5 was reduced to the amine 6-7, whichwas then immediately protected, such as reaction with4-monomethoxytritylchloride, to give the precursor 6-8 afterde-protection of 5′-OTBDPS group using TBAF/THF. The phosphitylation of6-9 using known protocols to give the desired 2′-O-methoxyethoxyadenine-NH-benzoyl phosphoramidite monomer 6-10.

Preparation of Intermediate (6-2):

To a solution of compound 1 (79.50 g, 210.68 mmol) in dry ACN (1.20 L)was added N-(5H-Purin-6-yl)benzamide (100.80 g, 421.36 mmol) and BSA(180.07 g, 884.86 mmol). The resulting suspension was stirred at 50° C.until clear. Then the mixture was cooled at −20° C. and TMSOTf (93.54 g,421.36 mmol) was added by syringe. Then the mixture was stirred at 70°C. for 72 h under N₂, and quenched with sat NaHCO₃ and extracted withDCM. The organic layer was dried over Na₂SO₄, then solvent wasevaporated, and the residue was purified on silica gel to affordcompound 6-2 (107.50 g, 192.26 mmol, 91.26% yield) as a yellow solid.¹H-NMR (400 MHz, DMSO): δ=11.28 (s, 1H), 8.64 (d, J=6.4 Hz, 2H), 8.05(d, J=8.0 Hz, 2H), 7.84 (d, J=8.0 Hz, 2H), 7.66 (t, J=7.6 Hz, 1H), 7.56(t, J=8.0 Hz, 2H), 7.33 (d, J=8.0 Hz, 2H), 6.37 (d, J=3.6 Hz, 1H), 6.17(dd, J=6.0 Hz, 1H), 5.09 (t, J=6.8 Hz, 1H), 4.69-4.56 (m, 2H), 4.40-4.38(m, 1H), 2.39 (s, 3H), 2.17 (s, 3H). ESI-LCMS: m/z 557.2 [M+H]⁺.

Preparation of Intermediate (6-3):

To a solution of compound 6-2 (107.50 g, 192.26 mmol) dissolved in 33wt. % methylamine in ethanol (600.00 mL), then the mixture were stirredat 20° C. for 16 h, then solvent was evaporated, washed with 50% EtOAcin petroleum ether (1.5 L), filtered to afford compound 6-3 (52.50 g,179.64 mmol, 93.44% yield) as a slightly yellow solid. ESI-LCMS: m/z293.1 [M+H]⁺.

Preparation of Intermediate (6-4):

A solution of compound 6-3 (52.50 g, 179.64 mmol), imidazole (18.32 g,269.46 mmol) and TBDPS-Cl (54.34 g, 197.60 mmol) in pyridine (500.00 mL)was stirred at 20° C. for 2 h, LC-MS showed 6-3 was consumed. Thenquenched with MeOH (30 mL), concentrated to give the crude product whichwas purified on silica gel with to afford compound 6-4 (72.60 g, 136.81mmol, 76.16% yield) as a white solid. ¹H-NMR (400 MHz, DMSO): δ=8.29 (s,1H), 8.10 (s, 1H), 7.63-7.59 (m, 4H), 7.48-7.33 (m, 8H), 6.36 (d, J=5.6Hz, 1H), 5.97 (d, J=4.4 Hz, 1H), 5.10-5.06 (m, 1H), 4.47 (t, J=5.6 Hz,1H), 4.14-4.11 (m, 1H), 3.94 (dd, J=11.2 Hz, 1H), 3.83 (dd, J=11.6 Hz,1H), 0.99 (s, 9H). ESI-LCMS: m/z 531.3 [M+H]⁺.

Preparation of Intermediate (6-5):

A solution of 6-4 (35.00 g, 65.96 mmol) and 1-Bromo-2-methoxyethane(18.33 g, 131.91 mmol) in dry DMF (400.00 mL), was added NaI (19.77 g,131.91 mmol) and Ag₂O (15.29 g, 65.96 mmol), the mixture was stirred atroom temperature for 5 h. Then the reaction was poured into ice water,extracted with EA, washed with brine and dried over anhydrous Na₂SO₄.The solvent was evaporated, and the residue was purified on silica gelto give 6-5 (23.70 g, 40.26 mmol, 61.04% yield) as a white solid andby-product of TBDPS lost 5.20 g, 9.81 mmol, 14.87% yield) as a whitesolid. ¹H-NMR (400 MHz, DMSO): δ=8.31 (s, 1H), 8.11 (s, 1H), 7.63-7.60(m, 4H), 7.47-7.44 (m, 2H), 7.40-7.36 (m, 6H), 6.10 (d, J=4.4 Hz, 1H),5.02 (t, J=4.8 Hz, 1H), 4.69 (t, J=5.6 Hz, 1H), 4.18-4.14 (m, 1H), 3.95(dd, J=11.6 Hz, 1H), 3.84 (dd, J=11.6 Hz, 1H), 3.78-3.75 (m, 2H), 3.45(t, J=4.8 Hz, 1H), 3.16 (s, 3H), 0.99 (s, 9H). ESI-LCMS: m/z 589.5[M+H]⁺.

Preparation of Intermediate (6-6):

To a solution of 6-5 (31.23 g, 53.04 mmol) in pyridine (300.00 mL) at 0°C., was added BzCl (11.22 g, 79.56 mmol) dropwise. The mixture wasstirred at r.t. for 2 h. Then the solution was cooled to 0° C., andammonium hydroxide (20 mL, 30%) was added and the mixture was allowed towarm to r.t., then the solvent was evaporated, 300 mL H₂O and 600 mL EAwere added into separate the solution, the aqueous was extracted by EA,combined the organic and washed with brine, dried over anhydrous Na₂SO₄,the solvent was removed and the residue was purified on silica gel togive 6-6 (28.70 g, 41.42 mmol, 78.09% yield) as a white solid. ESI-LCMS:m/z 693.4 [M+H]⁺.

Preparation of Intermediate (6-7):

A solution of 6-6 (28.70 g, 41.42 mmol) in EA (150.00 mL) was added Pd/C(3.00 g) and MeOH (150.00 mL) under H₂. The mixture was stirred at r.t.for 5 h. Then the reaction was filtered and the filtrate concentrated togive 6-7 (25.49 g, 38.22 mmol, 92.27% yield) as a gray solid. ESI-LCMS:m/z 667.3 [M+H]⁺.

Preparation of Intermediate (6-8):

To a solution of 6-7 (25.49 g, 38.22 mmol) and AgNO₃ (12.98 g, 76.44mmol) in DCM (300.00 mL) was added collidine (13.89 g, 114.66 mmol) andMMTrCl (19.43 g, 57.33 mmol), the mixture was stirred at r.t. for 2 h.Then the reaction was poured into ice water, the organic layer extractedwith DCM, washed with brine and dried over anhydrous Na₂SO₄, the solventwas removed and the residue was purified on silica gel to give 6-8(32.79 g, 34.92 mmol, 91.36% yield) as a gray solid.

Preparation of Intermediate (6-9):

A solution of 6-8 (32.79 g, 34.92 mmol) in THF (300.00 mL) was addedTBAF (1M, 35.00 mL), the mixture was stirred at room temperature for 15h. Then the solvent was removed and the residue was purified on silicagel with EA to give 6-9 (22.22 g, 31.71 mmol, 90.82% yield) as a whitesolid. ¹H-NMR (400 MHz, CDCl₃): δ=8.68 (s, 1H), 8.32 (s, 1H), 8.04 (d,J=7.2 Hz, 2H), 7.61-7.57 (m, 1H), 7.53-7.48 (m, 6H), 7.40 (d, J=8.8 Hz,2H), 7.21-7.12 (m, 6H), 6.73 (d, J=8.8 Hz, 2H), 6.09 (d, J=2.4 Hz, 2H),4.08-4.02 (m, 2H), 3.93-3.87 (m, 1H), 3.72 (s, 3H), 3.58-3.53 (m, 1H),3.43-3.39 (m, 3H), 3.24-3.19 (m, 4H), 2.19 (br, 1H).

Preparation of (6-10):

To a solution of 6-9 (14.00 g, 19.98 mmol), DMAP (488.19 mg, 4.00 mmol)and DIPEA (6.46 g, 49.95 mmol, 8.73 mL) in dry DCM (100.00 mL) was addedCEPCl (5.68 g, 23.98 mmol) dropwise under Ar. The mixture was stirred atroom temperature for 1 h. Then the reaction was washed with 10%NaHCO₃(aq) and brine, dried over Na₂SO₄, the solvent was removed and theresidue was purified by column chromatography, then concentrated to givethe crude product. The crude product (10 g, dissolved in 10 mL of ACN)was purified by Flash-Prep-HPLC to obtain 6-10 (12.60 g, 13.98 mmol,69.99% yield) as a white solid. Then the product was dissolved in drytoluene (15 mL) and concentrated three times, and with dry ACN threetimes. ¹H-NMR (400 MHz, CDCl₃): δ=9.12 (d, J=46.8 Hz, 1H), δ=8.71 (d,J=11.6 Hz, 1H), 8.50 (s, 0.6H), 8.22 (s, 0.4H), 8.04 (t, J=7.2 Hz, 2H),7.63-7.59 (m, 1H), 7.55-7.46 (m, 6H), 7.40-7.37 (m, 2H), 7.19-7.06 (m,6H), 6.69 (dd, J=8.8 Hz, 2H), 6.03 (d, J=3.2 Hz, 1H), 4.36-4.24 (m, 2H),3.92-3.78 (m, 2H), 3.71 (d, J=11.6 Hz, 3H), 3.67-3.33 (m, 7H), 3.29 (d,J=11.2 Hz, 3H), 3.17-3.10 (m, 1H), 2.88 (dd, J=27.2 Hz, 1H), 2.65-2.50(m, 2H), 2.38 (d, J=4.4 Hz, 0.4H), 1.80 (d, J=4.0 Hz, 0.6H), 1.23-1.15(m, 12H). ³¹PNMR (400 MHz, CDCl₃): 148.86, 148.22. ESI-LCMS: m/z 901.3[M+H]⁺.

Example 4

The synthesis of guanosine-based 2′-methoxyethoxy phosphoramidite 7-13shown in scheme 5. It was envisioned that stereo selective coupling ofC-3-azidoribose 1 with nucleobase 2-amino-6-chloropurine was key stepsto get various guanosine-based 3′-NH-MMTr-2′-O-alkylatedphosphoramidites. This was absolutely required to get efficient2′-O-alkylations and to eliminate any base alkylations. For thispurpose, C-3′-azido-chloro-amine purine nucleoside 7-2 b was synthesizedfrom nucleobase coupling of 1 and 2-amino-6-chloro purine in ˜70%isolated yield. Then, the protection of 2-amino group of 7-2 by usingMMTrCl to give 7-3, which after deprotection of 2′-O-acetyl and5′-O-toluoyl group by using NH₄OH/0° C. to give 2′-hydroxy intermediate7-4.

De-protection of 5′-O-toluyl group of intermediate 7-4 by treatment witha base followed by re-protection of 5′—OH group with trityl groupafforded key 2′-OH intermediate 9-6 for alkylation. This inventivede-protection and re-protection strategy was necessary for efficientsynthesis of 2′-O-alkylated intermediate 7-7 under basic conditions(NaHMDS/DMF).

Next hydrolysis of 6-methoxy group and de-tritylation simultaneouslyunder carefully controlled acidic conditions afforded3′-azido-2′-methoxy guanosine intermediate 7-8, which was subjected withiBuCl to give bisisobutyrate 7-9 followed by reduction and MMTrprotection of 3′-amino group of intermediate resulted in3′NH-MMTr-2′-methoxy ethoxy guanosine nucleoside 7-11. Further selectivede-protection of 5′-O-isobutyrate using basic conditions at lowtemperature resulted in 2′-methoxy ethoxy guanosine amidite precursor7-12. The phosphitylation of 7-12 using standard protocols to give thedesired 2′-O-methoxyethoxy guanosine phosphoramidite monomer 7-13.

Preparation of Intermediate (7-2):

To a solution of 1 (60.00 g, 152.00 mmol) in dry ACN (1300.00 mL) wasadded 2-amino-6-chloropurine (54.0 g, 318.5 mmol) and BSA (136.0 g,667.82 mmol). The resulting suspension was stirred at 50° C. untilclear. Then the mixture was cooled at −20° C. and TMSOTf (60.0 g, 271.00mmol) was added dropwise. Then the mixture was stirred at 70° C. for 18h, and quenched with sat NaHCO₃ and extracted with DCM. The organiclayer was dried over Na₂SO₄ and purified on silica gel andrecrystallized to give 7-2 (55.20 g, 113.38 mmol) as a white solid.¹H-NMR (400 MHz, CDCl₃): δ ppm 7.87 (d, J=8.4 Hz, 2H), 7.82 (s, 1H),7.25 (d, J=8.0 Hz, 2H), 6.09 (dd, J=5.6 Hz, 1H), 5.97 (d, J=4.0 Hz, 1H),5.17 (s, 2H), 4.84-4.80 (m, 2H), 4.60-4.56 (m, 1H), 4.44-4.41 (m, 1H),2.43 (s, 3H), 2.22 (s, 3H). ESI-LCMS: m/z 487.1 [M+H]⁺.

Preparation of Intermediate (7-3):

To a solution of 7-2 (33.2 g, 68.20 mmol) dissolved in DCM (300.00 mL),DMAP (1.67 g, 13.60 mmol) and DIEA (22.04 g, 136.4 mmol), MMTrCl (42.0g, 136.4 mmol) were added. And the mixture were stirred at r.t. for 16 hunder N₂. The mixture was then quenched with sat. NaHCO₃ and extractedwith DCM. The organic layer was dried over Na₂SO₄ and the residue waspurified by silica gel to afford compound 7-3 (51.00 g, 67.20 mmol,98.51% yield) as a white solid. ESI-LCMS: m/z 759.4 [M+H]⁺.

Preparation of Intermediate (7-4):

To a solution of 7-3 (54.00 g, 212.06 mmol) in THF (400 L) was addedNH₄OH (100.00 mL) at 0° C. The reaction mixture was stirred at r.t. for48 h, and concentrated and purified by silica gel with 1-2% MeOH in DCMto give 7-4 (51.00 g, 178.48 mmol, 99.98% yield) as a yellow solid.¹H-NMR (400 MHz, DMSO): δ ppm 8.26 (d, J=4.4 Hz, 1H), 8.17 (s, 1H),7.78-7.76 (m, 2H), 7.35-7.25 (m, 12H), 7.16 (t, J=7.2 Hz, 2H), 6.82 (dd,J=8.8 Hz, 2H), 6.14 (s, 1H), 5.62 (s, 1H), 4.40-4.35 (m, 4H), 3.69 (s,3H), 3.36 (s, 1H), 2.39 (s, 3H). ESI-LCMS: m/z 717.4 [M+H]⁺.

Preparation of Intermediate (7-5):

To a solution of sodium methoxide in methanol (300.00 mL, 2N) was added7-4 (47.00 g, 65.54 mmol), the mixture was stirred at r.t. for 1 h.

The mixture was concentrated in vacuo to give the crude product whichwas purified by CC (DCM:methyl alcohol=50:1) to give 7-5 (36.50 g, 61.38mmol, 93.66% yield) as white solid. ¹H-NMR (400 MHz, DMSO): δ ppm 8.15(s, 1H), 7.47 (s, 1H), 7.41-7.32 (m, 4H), 7.32-7.23 (m, 6H), 7.18 (t,J=7.16 Hz, 2H), 6.85 (d, J=8.68 Hz, 2H), 6.19 (s, 1H), 5.77 (s, 1H),5.28 (s, 1H), 4.81 (s, 1H), 4.20-4.06 (m, 1H), 3.90 (s, 1H), 3.72 (s,3H), 3.65-3.50 (m, 2H). ESI-LCMS: m/z 595.4 [M+H]⁺.

Preparation of Intermediate (7-6):

To a solution of 7-5 (36.50 g, 61.38 mmol) in dry DCM (5.00 mL) wasadded DIPEA (15.87 g, 122.77 mmol, 21.44 mL), DMAP (1.50 g, 12.28 mmol)and TrtCl (20.48 g, 73.66 mmol). The resulting suspension was stirred atr.t. for 20 h under Nitrogen and concentrated in vacuo. The product waspurified by cc (PE:EA=10:1˜3:1) to give 7-6 (46.00 g, 54.96 mmol, 89.55%yield) as white solid. ¹H-NMR (400 MHz, DMSO): δ ppm 8.08 (s, 1H),7.47-7.35 (m, 7H), 7.34-7.10 (m, 20H), 6.80 (d, J=7.76 Hz, 2H), 6.25 (s,1H), 5.81 (s, 1H), 4.86 (s, 1H) 4.10-3.87 (m, 2H), 3.69 (s, 3H),3.56-3.30 (m, 2H), 3.29-3.02 (m, 3H). 2.00 (s, 1H). ESI-LCMS: m/z 837.4[M+H]⁺.

Preparation of Intermediate (7-7):

To a solution of 7-6 (32.00 g, 38.23 mmol) in dry DMF (300.00 mL) wasadded tetrabutylammonium iodide (3.82 g, 11.47 mmol),1-bromo-2-methoxyethane (15.94 g, 114.69 mmol) and NaHMDS (21.03 g,114.69 mmol) added dropwise at −10° C. The resulting suspension wasstirred at −10° C. for 30 h and stirred at r.t. for 2 h. The product wasquenched with sat. NH₄Cl and extracted with DCM. The organic layer wasconcentrated in vacuo to give the crude product which was purified bysilica gel column to give 7-7 (31.46 g, 35.15 mmol, 91.94% yield) aswhite solid. ¹H-NMR (400 MHz, DMSO): δ ppm 8.04 (s, 1H), 7.40-7.34 (m,6H), 7.33-7.08 (m, 20H), 6.78 (d, J=8.9 Hz, 2H), 5.83 (s, 1H), 4.04-3.98(m, 1H), 3.68 (s, 3H), 3.67-3.46 (m, 3H), 3.44-3.36 (m, 2H), 3.27-3.15(m, 2H), 3.12 (s, 3H), 2.88 (s, 1H). ESI-LCMS: m/z 895.4 [M+H]⁺.

Preparation of Intermediate (7-8):

To a solution of 7-7 (42.00 g, 46.93 mmol) in 1,4-dioxane (150.00 mL)was added 6 N hydrochloric acid (46.93 mmol, 150.00 mL) at r.t. Theresulting suspension was stirred at r.t. for 30 min. The mixture wasextracted with petroleum ether. The water layer was stirred at r.t. for24 h. The mixture was concentrated in vacuo to give 7-8 (20.00 g, 37.12mmol,) as white solid. ¹H-NMR (400 MHz, DMSO): δ ppm 11.08-10.82 (m,1H), 8.41-8.13 (m, 1H), 7.35-6.99 (m, 1H), 6.85-6.62 (m, 2H), 5.90-5.78(m, 1H), 4.71 (t, J=5.16 Hz, 1H), 4.46 (t, J=4.64 Hz, 1H), 4.02-3.97 (m,1H), 3.78-3.70 (m, 2H), 3.70-3.62 (m, 1H), 3.57 (dd, J=12.12 Hz, 1H),3.43 (t, J=4.60 Hz, 2H), 3.18 (s, 3H). ESI-LCMS: m/z 367.2 [M+H]⁺.

Preparation of Intermediate (7-9):

To a solution of 7-8 (20.00 g, 54.60 mmol) in pyridine (100.00 mL) wasadded isobutyl chloride (17.45 g, 163.80 mmol) drop-wise at r.t. Theresulting suspension was stirred at r.t. for 1 h. The mixture wasextracted with DCM and washed with water. The organic layer wasconcentrated in vacuo. Dissolved in pyridine and added drop-wiseisobutyl chloride (17.45 g, 163.80 mmol) at r.t. The resultingsuspension was stirred at r.t. for 1 h., concentrated and purified bysilica gel column to give 7-9 (13.00 g, 25.67 mmol) as a white solid.ESI-LCMS: m/z 507.3 [M+H]⁺.

Preparation of Intermediate (7-10):

To a solution of 7-9 (13.00 g, 25.67 mmol) in THF (100.00 mL) was addedpalladium 10% on carbon (1.30 g), the mixture was stirred at r.t. for 20h at H₂. Filtered and the filtrate was concentrated in vacuo to give7-10 (11.70 g, 24.35 mmol) as brown solid. ESI-LCMS: m/z 481.3 [M+H]⁺.

Preparation of Intermediate (7-11):

To a solution of 7-10 (11.70 g, 24.35 mmol) in DCM (150.00 mL) was addedMMTrCl (11.25 g, 36.53 mmol) and 2,4,6-collidine (5.90 g, 48.70 mmol) at0° C., the mixture was stirred at r.t. for 30 min and added AgNO₃ (6.20g, 36.53 mmol) at 0° C. The mixture was stirred at r.t. for 2 h underN₂, filtered and the organic layer was washed by water and dried overNa₂SO₄, concentrated to give the crude product which was purified bysilica gel column to give 7-11 (8.5 g, 11.22 mmol) as a white solid.ESI-LCMS: m/z 753.5 [M+H]⁺.

Preparation of Intermediate (7-12):

To a solution of 7-11 (8.5 g, 11.29 mmol) in pyridine (50.00 mL) wasadded 2 N NaOH (50.00 mL) dropwise at 0° C., the mixture was stirred at0° C. for 20 min. Then the reaction was neutralized with saturated NH₄Cl(aq.) to pH=7-8, and 300 mL H₂O and 800 mL DCM were added in to separatethe solution, the aqueous was extracted by DCM, the combined organiclayer was washed with brine, dried over anhydrous Na₂SO₄, the solventwas removed and the residue was purified on silica gel to give 7-12 (6.3g, 9.23 mmol, 81.73% yield) as a white solid. ¹H-NMR (400 MHz, DMSO): δppm 11.78 (br s, 1H), 11.41 (s, 1H), 8.11 (s, 1H), 7.46 (t, J=7.28 Hz,4H), 7.31-7.21 (m, 6H), 7.15 (t, J=7.32 Hz, 2H), 6.76 (d, J=8.92 Hz,2H), 5.82 (s, 1H), 5.08 (t, J=4.08 Hz, 1H), 3.99-3.85 (m, 3H), 3.65 (s,3H), 3.62-3.55 (m, 1H), 3.42-3.38 (m, 2H), 3.29-3.19 (m, 1H), 3.10 (s,3H), 3.06-2.99 (m, 1H), 2.89-2.74 (m, 2H), 1.96 (d, J=4.08 Hz, 1H),1.20-1.12 (m, 6H). ESI-LCMS: m/z 883.4 [M+H]⁺.

Preparation of (7-13):

To a solution of 7-12 (6.3 g, 9.10 mmol) in DCM (60 mL) was added DMAP(222.00 mg) and DIPEA (5.9 mL). Then CEPCl (2.60 g) was added. Thereaction mixture was stirred at r.t. for 1 h., the mixture was washedwith saturated NaHCO₃ and brine, dried over Na₂SO₄, purified byFlash-Prep-HPLC. This resulted in 5.04 g 7-13 as a white solid. ¹H-NMR(400 MHz, CDCl₃): δ ppm 11.96 (br s, 1H), 8.57 (s, 1H), 7.98-7.67 (m,1H), 7.55-7.46 (m, 4H), 7.42-7.34 (m, 2H), 7.25-7.18 (m, 4H), 7.18-7.08(m, 2H), 6.78-6.68 (m, 2H), 5.79-5.69 (m, 1H), 4.27-4.11 (m, 2H),4.10-3.73 (m, 2H), 3.73-3.67 (m, 3H), 3.66-3.31 (m, 7H), 3.30-3.21 (m,3H), 2.07-2.97 (m, 1H), 2.94-2.78 (m, 1H), 2.70-2.50 (m, 3H), 2.30-1.68(m, 1H), 1.30-1.07 (m, 18H). ³¹P NMR (162 MHz, CDCl₃): 149.09, 148.09.ESI-LCMS: m/z 883.4 [M+H]⁺.

Example 5

The appropriately protected 2′-O-ethyl-3′-amino-5′-phosphoramidite(example 8, 9, 10, 11), were prepared after chemical transformationsshown in Schemes 6-10.

First for the synthesis of thymine based 3′-NH-MMtr-2′-O-ethylphosphoramidites example 9, intermediate 2 was protected such as methylpropyolate in the presence of dimethylaminopyridine (Scheme 6) to givebase N-3 protected intermediate 8-4 to facilitate the 2′-O-alkylation inhigher yield. Further deacetylation of 8-4 to give C-2′-hydroxyintermediate 8-5.

Further alkylation using iodoethane afforded 2′O-ethyl nucleoside 8-6.Intermediate 8-6 was converted to thymine base2′-O-ethyl-3′-amino-5′-phosphoramidite 8-11 by following the similarchemistry for compound 4-10 shown in previous Scheme 1.

Preparation of Intermediate (8-4):

To a solution of 8-2 (22.0 g, 49.62 mmol) in MeCN (400 mL) was addedDMAP (1.2 g, 9.92 mmol). Then 3 (5.8 g, 419.5 mmol) was added, themixture was stirred at r.t. for 2 h under N₂. Concentrated and purifiedby a silica gel column by (PE:EA=6:1) to afford 8-4 (22.0 g, 40.63 mmol,81.9% yield) as a yellow oil. ESI-LCMS: m/z 564 [M+Na]⁺.

Preparation of Intermediate (8-5):

To a solution of 8-4 (28.0 g, 51.71 mmol) in MeOH (400 mL) was addedcon. NH₄OH aqueous solution (28 mL) at 0° C. The reaction mixture wasstirred at 0° C. for 1.5 h. Concentrated and purified by a silica gelcolumn by (PE:EA=10:1˜2:1) to afford 8-5 (21.0 g, 42.04 mmol, 81.3%yield) as a yellow oil. ESI-LCMS: m/z 522 [M+Na]⁺.

Preparation of Intermediate (8-6):

To a solution of 8-5 (20.0 g, 40.04 mmol) in iodoethane (100 mL) wasadded Ag₂O (18.6 g, 80.08 mmol,). The reaction mixture was stirred at50° C. for 5 h, filtered with diatomite and concentrated to afford 8-6(16.0, 30.33 mmol, 75.7% yield) as a yellow oil which was used directlyin next step. ESI-LCMS: m/z 528 [M+H]⁺.

Preparation of Intermediate (8-7):

To a solution of 8-6 (16.0 g, 30.33 mmol) in MeCN (400 mL) was addedpyrrolidine (8.63 g, 121.32 mol, 12 mL), the reaction mixture wasstirred at r.t. overnight. Concentrated and purified by a silica gelcolumn by (DCM:MeOH=100:1˜50:1) to afford 7 (12.0 g, 27.94 mmol, 92.1%yield) as a yellow oil. ESI-LCMS: m/z 430 [M+H]⁺.

Preparation of Intermediate (8-8):

To a solution of 8-7 (12.0 g, 27.94 mmol) in THF (200 mL) was added Pd/C(1.2 g), the mixture was stirred at r.t. under H₂ overnight. LC-MSshowed 7 was totally consumed. Filtered and washed with DCM (100 mL*3),then concentrated to afford 8-8 (11.0 g, 27.27 mmol, 97.6% yield) as agray solid which was used directly in next step. ESI-LCMS: m/z 404[M+H]⁺.

Preparation of Intermediate (8-9):

To a solution of 8-8 (10.0 g, 24.79 mmol) in DCM (80 mL) was addedMMTrCl (11.4 g, 37.18 mmol), 2,4,6-collidine (2.0 g, 16.61 mmol, 6.5 mL)and AgNO₃ (6.3 g, 37.18 mmol), the mixture was stirred at r.t. for 1.5h. Filtered and the organic layer was washed with water and dried overNa₂SO₄, then concentrated and purified by a silica gel column by(PE:EA=5:1˜1:1) to afford 8-9 (16.0 g, 23.68 mmol, 95.5% yield) as alight-yellow solid.

Preparation of Intermediate (8-10):

8-9 (4.0 g, 5.92 mmol) was added to the solution of 1.0 N NaOH solution(20 mL, MeOH/H₂O=9:1). The reaction mixture was stirred at 40° C. for 2h, concentrated and extracted with DCM (20 mL*2), the organic layer wasdried over Na₂SO₄ and concentrated, the residue was purified by a silicagel column by (DCM:MeOH=200:1˜50:1) to afford 8-10 (3.0 g, 53.8 mmol,90.9 yield) as a white solid.

Preparation of (8-11):

To a solution of 8-10 (2.36 g, 4.23 mmol) in DCM (2.0 mL) was added DMAP(103 mg, 0.8 mmol) and DIPEA (2.2 g, 16.92 mmol, 2.96 mL). Then CEPCl(1.0 g, 4.23 mmol) was added. The reaction mixture was stirred at r.t.for 1 h., washed with saturated NaHCO₃(5 mL), separated the organiclayer and washed the water layer with DCM (10 mL*2). The combinedorganic layer was washed with brine, dried over Na₂SO₄, concentrated andpurified by Flash-Prep-HPLC to afford 8-11 (2.45 g, 3.23 mmol, 76.36%yield) as a white solid. ¹H NMR (400 MHz, CDCl₃) δ 8.62 (s, 1H), 7.74(dd, J=1.4 Hz, 0.5H), 7.60-7.50 (m, 4H), 7.51-7.41 (m, 2H), 7.34-7.16(m, 7H), 7.12 (d, J=1.4 Hz, 0.5H), 6.88-6.76 (m, 2H), 5.66 (s, 1H),4.37-4.23 (m, 1H), 4.16-4.05 (m, 1H), 4.05-3.94 (m, 0.5H), 3.88-3.74 (m,4.5H), 3.72-3.35 (m, 3H), 3.22 (td, J=10.3, 4.7 Hz, 0.5H), 3.03-2.89 (m,1.5H), 2.80-2.69 (m, 1H), 2.61 (t, J=6.5 Hz, 1H), 2.37 (td, J=6.6, 1.3Hz, 1H), 1.97 (d, J=3.5 Hz, 0.5H), 1.91 (dd, J=11.4, 1.2 Hz, 3H), 1.52(d, J=4.7 Hz, 0.5H), 1.29-1.17 (m, 12H), 1.08 (td, J=7.0, 4.9 Hz, 3H).³¹P NMR (162 MHz, CDCl₃) δ 149.31, 147.14. ESI-LCMS: m/z 576 [M+H]⁺.

Example 6

The 2′-O-ethyl-NH-benzoyl 5-methyl cytosine phosphoramidite 9-3 wasobtained by conversion of thymidine intermediate 8-8 into 3′-aminocytidine analogue 9-1 followed by phosphitylation using known protocolsto give the desired 2′-O-ethyl cytidine phosphoramidite monomer 9-3 asshown below in scheme 7.

Preparation of Intermediate (9-1):

To a solution of 8-8 (8.2 g, 12.09 mmol) in dry MeCN (40 mL) was addedTPSCl (2.5 g, 24.18 mmol) and DMAP (2.95 g, 24.18 mmol), then TEA (2.5g, 24.18 mmol, 3.4 mL) was added, the reaction mixture was stirred atr.t. for 12 h under N₂. Then con.NH₄OH aqueous solution (20 mL) wasadded, the result mixture was allowed to stirred at r.t. for another 3h, then concentrated and purified by silica gel column to afford 9-1(7.7 g, 11.35 mmol, 93.9% yield) as a light-yellow oil.

Preparation of Intermediate (9-2):

To a solution of 9-1 (8.0 g, 11.86 mmol) in pyridine (80 mL) was addedbenzoyl chloride (2.0 g, 14.23 mmol, 1.6 mL) at 0° C., the reactionmixture was allowed warm to r.t. and stirred for another 1 h, 2.0 N NaOH(80 mL, MeOH/H₂O=9:1) was added at 0° C., and then the mixture wasallowed to stirred at 0° C. for another 2 h, quenched with sat. NH₄Clsolution (20 mL) extracted with DCM (30 mL*2) dried over Na₂SO₄,filtered and concentrated. The residue was purified by a silica gelchromatography (DCM:MeOH=200:1˜50:1) to afford 9-2 (5.4 g, 8.2 mmol,66.3% yield) as yellow solid. ¹H NMR (400 MHz, CDCl₃) δ 13.35 (s, 1H),8.40-8.28 (m, 2H), 7.93 (d, J=1.3 Hz, 1H), 7.61-7.51 (m, 5H), 7.51-7.43(m, 4H), 7.33-7.22 (m, 6H), 6.89-6.72 (m, 2H), 5.66 (s, 1H), 4.26-4.19(m, 2H), 4.08 (dt, J=10.4, 2.0 Hz, 1H), 3.80 (s, 3H), 3.74-3.63 (m, 1H),3.12 (td, J=10.3, 4.5 Hz, 1H), 2.95-2.84 (m, 2H), 2.05 (d, J=1.1 Hz,3H), 1.91 (d, J=4.5 Hz, 1H), 1.29 (t, J=7.1 Hz, 1H) 1.13 (t, J=7.0 Hz,3H).

Preparation of (9-3):

To a solution of 9-2 (4.2 g, 6.36 mmol) in DCM (4 mL) was added DMAP(776 mg, 6.36 mmol), DIPEA (821 mg, 6.36 mmol, 1.1 mL) and then CEPCl(1.5 g, 6.36 mmol). The reaction mixture was stirred at r.t. for 1 h.,quenched with sat. NaHCO₃ solution, extracted with DCM (30 mL*2), theorganic layer was dried over Na₂SO₄ and the solvent was evaporated, theresidue was purified by a Flash-Prep-HPLC to afford 9-3 (4.9 g, 5.69mmol, 89.47% yield) as light yellow solid. ¹H NMR (400 MHz, CDCl₃) δ13.31 (s, 1H), 8.40-8.31 (m, 2H), 8.03 (s, 0.5H), 7.60-7.52 (m, 5H),7.52-7.43 (m, 4H), 7.40 (s, 0.5H), 7.31-7.18 (m, 7H), 6.86-6.78 (m, 2H),5.67 (d, J=2.3 Hz, 1H), 4.42-4.26 (m, 1H), 4.24-4.11 (m, 1H), 4.06-3.98(m, 0.5H), 3.90-3.81 (m, 1.5H), 3.79 (d, J=3.0 Hz, 3H), 3.75-3.56 (m,2H), 3.55-3.37 (m, 1H), 3.22 (td, J=10.4, 4.5 Hz, 0.5H), 3.04-2.87 (m,1.5H), 2.86-2.73 (m, 1H), 2.63 (t, J=6.4 Hz, 1H), 2.40 (t, J=6.5 Hz,1H), 2.13 (dd, J=19.9, 1.1 Hz, 3H), 1.99 (d, J=3.8 Hz, 0.5H), 1.54 (d,J=4.5 Hz, 0.5H), 1.31-1.21 (m, 12H), 1.10 (td, J=7.0, 4.8 Hz, 3H). ³¹PNMR (162 MHz, CDCl₃) δ 149.48, 147.08. ESI-LCMS: m/z 861 [M+H]⁺.

Example 7

The synthesis of the 2′-O-ethyl adenosine phosphoramidite 10-8 wasachieved as shown below in scheme 8. Protection of exocyclic amine groupof 6-4 with bulky protecting group such as 4-monomethoxy trityl groupwas necessary to achieve 2′-O-alkylation in high yield. Then,intermediate 6-4 by reaction with Ag₂O/EtI afforded 2′-O-ethylintermediate 10-2 in 94% isolated yield after deprotection of MMTrgroup-. This protection strategy resulted in efficient synthesis of key3′-azido-2′-OEt intermediate 10-3 in high yields. This intermediate 10-3was converted to 2′-O-ethyl adenosine phosphoramidite 10-8 by followingthe similar chemistry described for compound 6-10 (scheme 4).

Preparation of Intermediate (10-1):

To a solution of compound 6-4 (23.6 g, 44 mmol) in DCM (400.00 mL), DMAP(543 mg, 4 mmol) and TEA (9.2 g, 9 mmol), MMTr-Cl (20.6 g, 67 mmol) wasadded, the mixture were stirred at 20° C. for 16 h under N₂. LC-MSshowed 6-4 was consumed, quenched with sat. NaHCO₃ and extracted withDCM. The organic layer was dried over Na₂SO₄ and concentrated to givethe crude product which was purified on silica gel with 20-50% EtOAc inpetroleum ether to afford compound 10-1 (26.1 g, 32 mmol, 73.09% yield)as a slightly yellow solid. ¹H-NMR (400 MHz, DMSO): δ=8.38 (s, 1H), 7.84(s, 1H), 7.60-7.55 (m, 4H), 7.46-7.20 (m, 19H), 6.84 (d, J 8.8 Hz, 2H),6.35 (d, J 5.6 Hz, 1H), 5.95 (d, J 4.4 Hz, 1H), 5.14-5.12 (m, 1H), 4.46(t, J 5.6 Hz, 2H), 4.11-4.05 (m, 1H), 3.90-3.82 (m, 2H), 3.72 (s, 3H),0.94 (s, 8H). ESI-LCMS: m/z 803.4 [M+H]⁺.

Preparation of Intermediate (10-2):

To a solution of compound 10-1 (26.1 g, 32 mmol) in CH₃CH₂I (200 mL),Ag₂O (9.7 g, 42 mmol) was added. The mixture was refluxed at 80° C. for1 h. Then filtered to get the compound 10-2 (25.4 g, 30 mmol, 94.04%yield) as a yellow solid. ESI-LCMS: m/z 831.3 [M+H]⁺.

Preparation of Intermediate (10-3):

A solution of compound 10-2 (25.4 g, 30 mmol) was dissolved in DCM (200mL) was added TCA (12 mL), the mixture was stirred at 20° C. for 6 h,LC-MS showed 10-2 was consumed, then washed with saturated NaHCO₃,concentrated to give the crude product which was purified on silica gelwith 1-3% MeOH in DCM to afford compound 10-3 (15.7 g, 28 mmol, 91.95%yield) as a white solid. ¹H-NMR (400 MHz, DMSO): δ=8.33 (s, 1H), 8.10(s, 1H), 7.63-7.60 (m, 4H), 7.47-7.37 (m, 8H), 6.09 (d, J 4.0 Hz, 1H),4.95 (t, J 4.8 Hz, 1H), 4.67 (t, J 5.6 Hz, 1H), 4.17-4.13 (m, 1H),3.97-3.93 (m, 1H), 3.86-3.82 (m, 1H), 3.68-3.63 (m, 2H), 1.15-1.11 (m,3H), 0.97 (s, 9H). ESI-LCMS: m/z 589.2 [M+H]⁺.

Preparation of Intermediate (10-4):

To a solution of compound 10-3 (15.7 g, 28 mmol) in pyridine (150 mL) at0° C., BzCl (4.7 g, 34 mmol) was added by syringe over 5 minutes, thenthe mixture was allowed to warm up to 20° C. Then stirred at r.t. underN₂ for 1 h. The solution was cooled to 0° C., and ammonium hydroxide (20mL, 30%) was added and the mixture was allowed to warm to r.t. andstirred at r.t. for 2 h. The mixture was diluted with EA and Water,extracted with EA, the combined organic layer was washed with brine,dried over anhydrous Na₂SO₄, concentrated to give the crude productwhich was purified on silica gel to afford compound 10-4(10.9 g, 16mmol, 58.52% yield) as a white solid. ESI-LCMS: m/z 663.8 [M+H]⁺.

Preparation of Intermediate (10-5):

To a solution of compound 10-4 (10.9 g, 16 mmol) in THF (100 mL), Pd/C(1.9 g) were added, the mixture was stirred at 20° C. for 6 h under H₂,then filtered and the filtrate concentrated to afford compound 10-5 (9.8g, 15 mmol, 93.95% yield) as a white solid. ESI-LCMS: m/z 637.4 [M+H]⁺.

Preparation of Intermediate (10-6):

To a solution of compound 10-5 (9.8 g, 15 mmol) in anhydrous DCM (100mL), collidine (3.7 g, 31 mmol) and AgNO₃ (3.9 g, 31 mmol), MMTr-Cl (5.7g, 18 mmol) were added, the mixture was stirred at 20° C. for 1 h underN₂, then filtered, washed with H₂O and dried over Na₂SO₄, concentratedto give the residue which was purified on silica gel with 20-50% EA inpetroleum ether to afford compound 10-6 (10.5 g, 11 mmol, 75.19% yield)as a white solid.

Preparation of Intermediate (10-7):

To a solution of compound 10-6 (10.5 g, 11 mmol) in THF (100 mL), TBAF(1M) (11 mmol, 12 mL) was added, the mixture was stirred at 20° C.overnight, then washed with saturated NaHCO₃, concentrated to give theresidue which was purified on silica gel with 1-2% MeOH in DCM to affordcompound 10-7 (5.6 g, 8 mmol, 72.28% yield) as a white solid.

Preparation of (10-8):

To a solution of compound 10-7 (5.6 g, 8 mmol) in DCM (50 mL) was addedDMAP (204 mg, 2 mmol) and DIPEA (3.2 g, 25 mmol, 4.3 mL), CEPCl (2.3 g,10 mmol) was added. The reaction mixture was stirred at r.t. for 1 h.TLC showed 10-7 was consumed, washed with saturated NaHCO₃ and brine,dried over Na₂SO₄, concentrated to give the crude product which waspurified by column chromatography and Flash-Prep-HPLC to give 10-8 (5.30g, 6.09 mmol, 72.88% yield) as a white solid. ¹H-NMR (400 MHz, CDCl₃):δ=9.17 (s, 0.5H), 9.07 (s, 0.5H), 8.75 (s, 0.5H), 8.72 (s, 0.5H), 8.51(s, 0.5H), 8.22 (s, 0.5H), 8.05 (t, J 7.2 Hz, 2H), 7.65-7.61 (m, 1H),7.57-7.48 (m, 6H), 7.39 (dd, J 8.8 Hz, 2H), 7.21-7.10 (m, 6H), 6.74-6.69(m, 2H), 6.01 (d, J 3.9 Hz, 1H), 4.40-4.21 (m, 2H), 4.14-4.11 (m, 0.5H),3.99-3.93 (m, 0.5H), 3.88-3.80 (m, 1H), 3.70-3.50 (m, 7H), 3.41-3.32 (m,1H), 2.93-2.80 (m, 2H), 2.68-2.50 (m, 2H), 2.27 (d, J 4.0 Hz, 0.5H),2.27 (d, J 4.0 Hz, 0.5H), 1.25-1.12 (m, 15H). ³¹P NMR (162 MHz, CDCl₃):148.79, 148.08. ESI-LCMS: m/z 870.3[M+H]⁺.

Example 8

The synthesis of guanosine-based 2′-O-ethyl phosphoramidite 12-9 shownbelow in scheme 9 starting from key 2′-hydroxy intermediate 7-4.Intermediate 7-4 was subjected to 2′-O-alkylation using EtI/Ag₂O orEt//K₂CO₃ desired 2′-OEt intermediate 7-5 could not be obtained in highyield as shown below in scheme 9.

Therefore, an innovative way to improve on 2′-O alkylation and to makemore stable guanosine intermediate under acidic conditions was designedas shown below in scheme 10. The reaction of 6-chloro intermediate 7-4with NaH/BnOH/THF resulted in simultaneous substitution of C-6-Cl withBnOH and hydrolysis of 5′-O-toluoylester in one step to give C-6-OBnintermediate 12-1.

The 5′-hydroxy group was selectively protected with DMTr to give 2′-OHnucleoside 12-2. Resulting intermediate 12-2 was reacted with NaH/EtI togive 2′-O-ethyl intermediate 12-3, which was subjected under acidicconditions (TFA/DCM) to afford 5′-hydroxy intermediate 12-4 without anyde-purination. Then, the reaction of 12-4 with iBuCl resulted inbis-isobutyrate intermediate 12-5, which on hydrogenation by using Pd/Cgave 2′-O-ethyl guanosine intermediate 12-6. This de-protection andre-protection strategy were applied successfully for efficient2′-O-alkylations under basic conditions (NaH/EtI/THF). The resulted2′-O-ethyl intermediate 12-6 was converted to desired2′-O-ethylguanosine phosphoramidite monomer 12-9 by following thesimilar procedure described for compound 7-13 in example 12.

Preparation of Intermediate (12-1):

To a solution of compound 7-4 (48.0 g, 66 mmol) in 50 mL of THF wasadded 90 mL of a sodium benzyloxide solution in benzyl alcohol (freshlyprepared by addition of NaH(13 g) in 90 mL of benzyl alcohol). Thereaction mixture was stirred at room temperature for 1 h and treatedwith 50 mL of a saturated solution of NH₄C₁. The mixture was extractedwith CH₂Cl₂ and the combined organic layers dried over Na₂SO₄. The crudeproduct was purified by column chromatography to give 12-1 (17.5 g, 26mmol, 45.63%). ¹H-NMR (400 MHz, DMSO): δ ppm 8.13 (s, 1H), 7.59 (s, 1H),7.34-7.16 (m, 15H), 6.82 (d, J=8.8 Hz, 2H), 6.13 (s, 1H), 5.76 (s, 1H),5.20-5.12 (m, 1H), 5.10-4.78 (m, 2H), 3.88-3.70 (m, 2H), 3.60 (s, 3H),3.58-3.51 (m, 1H), 3.34 (s, 1H). ESI-LCMS: m/z 671.2 [M+H]⁺.

Preparation of Intermediate (12-2):

To a solution of compound 12-1 (17.5 g, 26 mmol) in pyridine (200 mL)was added DMTrCl (10.5 g, 31 mmol) at 0° C. Then the mixture was stirredat 35° C. for 4 h. Solvent was evaporated. The crude product waspurified by column chromatography to give product 12-2 (21.8 g, 22 mmol,85.87% yield) as a white solid. ESI-LCMS: m/z 974.6 [M+H]⁺.

Preparation of Intermediate (12-3):

To a solution of 12-2 (21.8 g, 22 mmol) in THF (300 mL) was added NaH(1.1 g, 44 mmol) at 0° C. Then EtI (10.4 g, 67 mmol) was added inreaction mixture and the mixture was stirred at 35° C. for 4 h, thentreated with 50 mL of a saturated solution of NH₄C₁. The mixture wasextracted with CH₂Cl₂ and the combined organic layers dried over Na₂SO₄.Solvent was evaporated to give 12-3 (21.2 g, 21 mmol, 94.55% yield) as ayellow solid.

Preparation of Intermediate (12-4):

To a solution of 12-3 (21.2 g, 21 mmol) in DCM (200 mL) was added TFA(10 mL) at 0° C. Then the mixture was stirred at 35° C. for 4 h.Quenched with saturated NaHCO₃ and washed with brine, dried over Na₂SO₄,purified by purified by column chromatography to give 12-4 (8.7 g, 20mmol, 96.32% yield) as a white solid. ¹H-NMR (400 MHz, DMSO): δ ppm 8.17(s, 1H), 7.53-7.50 (m, 2H), 7.42-7.36 (m, 3H), 6.61 (d, J=8.8 Hz, 2H),5.91-5.90 (m, 1H), 5.76 (s, 1H), 5.51-5.50 (m, 2H), 4.76-4.75 (m, 1H),4.55-4.54 (m, 1H), 4.06-3.97 (m, 3H), 3.64-3.58 (m, 4H), 2.51 (s, 1H).ESI-LCMS: m/z 427.2 [M+H]⁺.

Preparation of Intermediate (12-5):

To a solution of 12-4 (8.7 g, 20 mmol) in pyridine (100 mL) was addediBuCl (5.4 g, 51 mmol), the mixture was stirred at r.t. for 1.5 h. Theorganic layer was washed by water and dried over Na₂SO₄ and purified bycolumn chromatography to give 12-5 (8.5 g, 15 mmol, 73.54% yield) as awhite solid. ESI-LCMS: m/z 567.4 [M+H]⁺.

Preparation of Intermediate (12-6):

To a solution of 12-5 (8.5 g, 15 mmol) in THF (100 mL) was added Pd/C(801 mg, 6 mmol) at 25° C. Then the mixture was stirred under H₂ for 16h. Filtered and washed by EA, concentrated to give crude product 12-6(6.7 g, 14 mmol, 99.15% yield) as a yellow solid. ESI-LCMS: m/z 451.3[M+H]⁺.

Preparation of Intermediate (12-7): To a solution of compound 12-6 (6.7g, 14 mmol) in anhydrous DCM (70 mL), collidine (3.7 g, 31 mmol) andAgNO₃ (3.0 g, 17 mmol), MMTr-Cl (5.5 g, 17 mmol) were added, the mixturewas stirred at 20° C. for 1 h under N₂, then filtered, washed with H₂Oand dried over Na₂SO₄, concentrated to give the residue which waspurified by column chromatography to afford compound 12-7 (8.4 g, 11mmol, 75.19% yield) as a white solid.

Preparation of Intermediate (12-8):

Compound 12-7 (8.4 g, 11 mmol) was added to 80 mL of 1 N NaOH solutionin MeOH/THF/H₂O (65/30/5) at 0° C. The suspension was stirred at 0° C.for 2 h. The reaction was quenched by addition of sat. NH₄Cl solution.The solution was extracted with DCM and the combined organic layers werewashed with sat. NaHCO₃ solution, brine, dried over Na₂SO₄, filtered,and concentrated. The residue was purified by column chromatography togive 12-8 (5.60 g, 8.35 mmol, 72.28% yield) as white solid. ¹H-NMR (400MHz, DMSO): δ ppm 12.13 (br s, 1H), 11.38 (s, 1H), 8.10 (s, 1H),7.47-7.44 (m, 4H), 7.30-7.13 (m, 8H), 6.75 (d, J=8.96 Hz, 2H), 5.79 (s,1H), 5.10 (t, J=4.28 Hz, 1H), 3.95-3.89 (m, 3H), 3.65 (s, 3H), 3.46-3.26(m, 2H), 2.84-2.80 (m, 2H), 2.79-2.72 (m, 1H), 1.85 (s, 1H), 1.18-1.13(m, 6H). 1.08-0.98 (m, 3H).

Preparation of (12-9):

To a solution of 12-8 (5.60 g, 8.35 mmol) in DCM (50 mL) was added DMAP(220 mg) and DIPEA (3 mL). Then CEPCl (2.6 g) was added. The reactionmixture was stirred at r.t. for 1 h, washed with saturated NaHCO₃ andbrine, dried over Na₂SO₄, purified by column chromatography andFlash-Prep-HPLC. This resulted in 5.4 g (70.04%) of 12-9 as a whitesolid. ¹H-NMR (400 MHz, CDCl₃): δ ppm 12.11 (br s, 1H), 8.88 (s, 1H),7.97-7.64 (m, 1H), 7.51-7.49 (m, 4H), 7.39-7.34 (m, 2H), 7.23-7.09 (m,6H), 6.75-6.69 (m, 2H), 5.67-5.65 (m, 1H), 4.22-4.08 (m, 2H), 3.84-3.29(m, 10H), 2.87-2.51 (m, 5H), 2.08-1.61 (m, 1H), 1.24-1.07 (m, 18H),1.01-0.98 (m, 3H). ³¹PNMR (162 MHz, CDCl₃): 149.03, 147.96. ESI-LCMS:m/z 853.4 [M+H]⁺.

Example 9

Preparation of Intermediate (13-3):

A solution of 13-2 (260.0 g, 605.5 mmol) and DBU (305.0 g, 1.21 mol) inDMF (1.5 L) was added PMBCl (113.7 g, 908.2 mmol). The mixture wasstirred at r.t. for 2 h, poured into cold water, extracted with EA,washed with brine and dried over anhydrous Na₂SO₄, concentrated to givethe crude product 13-3 (460.0 g, crude) as a yellow oil. ESI-LCMS: m/z550 [M+H]⁺.

Preparation of Intermediate (13-4):

A solution of 13-3 (460.0 g, crude) in the mixture of THF (1.5 L) andH₂O (500 mL) was added NaOH (167.4 g, 4.1 mol). The reaction was stirredat under N₂. The mixture was stirred at room temperature for 1 h,quenched with MeOH, concentrated to obtain a residue which was purifiedby column chromatography to give 13-5 (330.0 g, 477.0 mmol, 89.7% yield)as a yellow solid. ¹H-NMR (400 MHz, DMSO-d₆): δ=7.81 (d, J=8.0 Hz, 1H),7.39 (d, J=7.6 Hz, 2H), 7.33 (t, J=7.6 Hz, 2H), 7.28-7.25 (m, 7H), 6.91(d, J=7.6 Hz, 4H), 6.86 (d, J=8.4 Hz, 2H), 6.33 (d, J=5.2 Hz, 1H,exchanged with D₂O), 5.49 (d, J=8.0 Hz, 1H), 4.91 (s, 2H), 4.51 (dd,J=5.2, 8.8 Hz, 1H), 4.21 (t, J=6.8 Hz, 1H), 4.08-4.05 (m, 1H), 3.75 (s,6H), 3.72 (s, 3H). ESI-LCMS: m/z 692 [M+H]⁺.

Preparation of Intermediate (13-6):

A solution of 13-5 (150.0 g, 216.8 mmol) in dry THF (750 mL) were addedNaH (10.4 g, 433.7 mmol) and Methyl p-toluenesulfonate (66.1 g, 433.7mmol) at 0° C., the mixture was allowed to stir at r.t., and TBAI (16.0g, 43.3 mmol) was added. The reaction was stirred at 80° C. for 2 days,and then quenched with sat NH₄Cl(aq.), extracted with EA, washed withbrine, dried over Na₂SO₄, concentrated to obtain a residue which waspurified by column chromatography to give 13-6 (55.0 g, 70.0 mmol,32.28% yield) as a yellow solid. ESI-LCMS: m/z 786 [M+H]⁺.

Preparation of Intermediate (13-7):

A solution of 13-6 (52.0 g, 66.1 mmol) and K₂CO₃ (18.2 g, 132.3 mmol) indry DMF (500 mL) was added Methyl p-toluenesulfonate (18.4 g, 99.2 mmol)under Ar. The mixture was stirred at r.t. for 15 h, poured into coldwater, extracted with EA, washed with brine and dried over anhydrousNa₂SO₄, concentrated to obtain a residue which was purified by columnchromatography to give 7 (38.5 g, 48.1 mmol, 72.74% yield) as a whitesolid. ¹H-NMR (400 MHz, DMSO-d₆): δ=7.81 (d, J=8.4 Hz, 1H), 7.41-7.38(m, 2H), 7.34-7.31 (m, 2H), 7.28-7.23 (m, 7H), 6.92-6.90 (m, 4H),6.84-6.81 (m, 2H), 5.98 (d, J=3.6 Hz, 1H), 5.59 (d, J=8.4 Hz, 1H), 5.40(dd, J=3.6, 6.4 Hz, 1H), 4.90 (dd, J=14.0, 32.0 Hz, 2H), 4.67 (dd,J=6.4, 7.6 Hz, 1H), 4.09-4.05 (m, 1H), 3.74 (d, J=0.8 Hz, 6H), 3.70 (s,3H). ¹⁹F-NMR (376 MHz, DMSO-d6): −75.31, −75.69, −76.69, −77.35.ESI-LCMS: m/z 800 [M+H]+.

Preparation of Intermediate (13-8):

To a solution of 13-7 (33.0 g, 41.2 mmol) in THF (600 mL) were addedNaBH₄ (2.3 g, 61.8 mmol) and EtOH (20 mL) at 0° C. The mixture wasallowed to stir at r.t. for 4 h, and quenched with sat NH₄Cl(aq.),extracted with EA, washed with brine, dried over Na₂SO₄, concentrated toobtain a residue which was purified by column chromatography to give13-8 (25.0 g, 32.3 mmol, 78.51% yield) as a white solid. ¹H-NMR (400MHz, DMSO-d₆): δ=7.78 (d, J=8.4 Hz, 1H), 7.41-7.38 (m, 2H), 7.34-7.30(m, 2H), 7.28-7.21 (m, 7H), 6.92-6.89 (m, 4H), 6.84-6.82 (m, 2H), 5.98(d, J=4.4 Hz, 1H), 5.83 (t, J=6.8 Hz, 1H), 5.60 (d, J=8.0 Hz, 1H), 5.30(t, J=5.3 Hz, 1H), 4.90 (dd, J=14.0, 33.2 Hz, 2H), 4.61 (t, J=6.0 Hz,1H), 4.08-4.05 (m, 1H), 3.74 (d, J=0.8 Hz, 6H), 3.70 (s, 3H), 3.39 (dd,J=4.0, 14.8 Hz, 1H), 3.30 (dd, J=2.8, 11.2 Hz, 1H). ¹⁹F-NMR (376 MHz,DMSO-d6): −79.67, −80.04, −80.87, −81.25. ESI-LCMS: m/z 772 [M+H]⁺.

Preparation of Intermediate (13-9):

To a solution of 13-8 (26.0 g, 33.6 mmol) in THF (250 mL) were added NaH(808 mg, 33.6 mmol) and MeI (23.9 g, 168.4 mmol, 10.4 mL) at 0° C.

The mixture was allowed to stir at r.t. for 15 h, and poured into coldwater, extracted with EA, washed with brine, dried over Na₂SO₄,concentrated to obtain the crude product 13-9 (29.0 g) as a yellow oil.ESI-LCMS: m/z 786 [M+H]+.

Preparation of Intermediate (13-10):

A solution of 13-9 (29.0 g, 36.9 mmol) in DCM (200 mL) was added asolution of p-TsOH (10.0 g) in MeOH (20 mL) The mixture was stirred atr.t. for 0.5 h, and washed with saturated NaHCO₃ to pH=7, dried overNa₂SO₄, concentrated to obtain a residue which was purified by columnchromatography to give 13-10 (13.5 g, 27.9 mmol, 82.90% yield over twosteps). ¹H-NMR (400 MHz, DMSO-d₆): δ=7.99 (d, J=4.4 Hz, 1H), 7.22 (d,J=8.4 Hz, 2H), 6.85 (d, J=8.8 Hz, 2H), 6.01 (d, J=5.2 Hz, 1H), 5.89 (d,J=8.0 Hz, 1H), 5.46 (t, J=4.8 Hz, 1H), 5.17 (t, J=6.4 Hz, 1H), 4.90 (dd,J=14.0, 32.4 Hz, 2H), 4.43 (t, J=5.2 Hz, 1H), 4.00-3.97 (m, 1H),3.74-3.67 (m, 6H), 3.64-3.60 (m, 1H), 3.33 (s, 3H). ESI-LCMS: m/z 484[M+H]⁺.

Preparation of Intermediate (13-11):

To a solution of 13-10 (28.0 g, 57.9 mmol) and TEA (11.7 g, 115.8 mmol,16 mL) in DCM (300 mL) was added BzCl (12.2 g, 86.8 mmol) under Ar. Themixture was stirred at r.t. for 2 h, and poured into cold water,extracted with EA, washed with brine, dried over Na₂SO₄, concentrated toobtain a residue which was purified by column chromatography to give13-11 (31.0 g, 52.7 mmol, 91.10% yield) as a clear oil. ¹H-NMR (400 MHz,DMSO-d₆): δ=8.00 (d, J=3.2 Hz, 1H), 7.77 (d, J=8.0 Hz, 1H), 7.68 (t,J=7.6 Hz, 1H), 7.54 (t, J=7.6 Hz, 1H), 7.20 (d, J=8.8 Hz, 2H), 6.82 (d,J=8.4 Hz, 2H), 5.97 (d, J=4.4 Hz, 1H), 5.78 (d, J=8.4 Hz, 1H), 5.38 (t,J=6.4 Hz, 1H), 4.87 (dd, J=14.0, 27.2 Hz, 2H), 4.65-4.60 (m, 2H),4.56-4.51 (m, 1H), 4.28-4.24 (m, 1H), 3.76 (t, J=10.4 Hz, 2H), 3.69 (s,3H), 3.35 (s, 3H). ESI-LCMS: m/z 588 [M+H]⁺.

Preparation of Intermediate (13-12):

A solution of 13-11 (30.0 g, 51.0 mmol) in the mixture of ACN (300 mL)and H₂O (100 mL) was added CAN (83.9 g, 153.1 mmol). After the mixturewas stirred at 45° C. for 24 h, poured into water, extracted with EA,washed with brine and dried over anhydrous Na₂SO₄, concentrated toobtain a residue which was purified by column chromatography to give13-12 (20.0 g, 42.7 mmol, 83.80% yield) as a white solid. ESI-LCMS: m/z468 [M+H]⁺.

Preparation of Intermediate (13-13):

A solution of 13-12 (13.0 g, 27.8 mmol) in THF (150 mL) were added TPP(10.9 g, 41.7 mmol) and H₂O (5.0 g, 278.1 mmol). The mixture was stirredat 60° C. for 5 h, concentrated to obtain a residue which was purifiedby column chromatography to give 13-13 (11.5 g, 26.0 mmol, 93.67% yield)as a white solid. ESI-LCMS: m/z 442 [M+H]⁺.

Preparation of Intermediate (13-14):

A solution of 13-13 (10.5 g, 23.7 mmol) and DMAP (581 mg, 4.7 mmol) inDCM (100 mL) were added MMTrCl (11.0 g, 35.6 mmol) and TEA (4.8 g, 47.5mmol, 6.6 mL) under N₂. The mixture was stirred at r.t. for 1 h,quenched with MeOH and concentrated to obtain a residue which waspurified by column chromatography to give 13-14 (15.7 g, 22.0 mmol,92.47% yield) as a white solid. ESI-LCMS: m/z 714 [M+H]⁺.

Preparation of Intermediate (13-15):

A solution of 13-14 (4.0 g, 5.6 mmol) in 1 N NaOH(MeOH:H2O=4:1) (80 mL),the mixture was stirred at room temperature for 1 h, and the reactionwas neutralized with saturated NH₄Cl (aq) to pH=7˜8, extracted by EA,washed with brine, dried over anhydrous Na₂SO₄, concentrated to obtain aresidue which was purified by column chromatography to give 13-15 (3.2g, 5.2 mmol, 93.66% yield) as a white solid. ¹H-NMR (400 MHz, DMSO-d₆):δ=11.37 (d, J=2.4 Hz, 1H, exchanged with D₂O), 7.91 (d, J=8.0 Hz, 1H),7.50-7.48 (m, 4H), 7.39-7.36 (m, 2H), 7.29 (t, J=7.6 Hz, 4H), 7.22-7.18(m, 2H), 6.86 (d, J=8.8 Hz, 2H), 6.19 (d, J=6.0 Hz, 1H), 5.67 (dd,J=2.0, 8.0 Hz, 1H), 5.16 (t, J=4.8 Hz, 1H), 4.28 (t, J=5.6 Hz, 1H), 3.78(dd, J=7.6, 12.8 Hz, 2H), 3.73 (s, 3H), 3.38 (s, 3H), 3.31-3.26 (m, 4H),3.06-3.01 (m, 1H). ¹⁹F-NMR (376 MHz, DMSO-d₆): −73.96, −74.34, −78.81,−79.20. ESI-LCMS: m/z 610 [M+H]⁺.

Preparation of (13-16):

A solution of 13-15 (1.5 g, 2.4 mmol) and DCI (145 mg, 1.2 mmol) in dryDCM (30 mL) was added CEP[N(iPr)₂]₂ (816 mg, 2.7 mmol) under Ar. Themixture was stirred at r.t. for 1 h. The reaction was washed with 10%NaHCO₃(aq.) and brine, dried over Na₂SO₄ and concentrated to obtain thecrude product which was purified by column chromatography. This resultedin to give 13-16 (1.5 g, 1.8 mmol, 75.28% yield) as a white solid.¹H-NMR (400 MHz, DMSO-d₆): δ=11.41 (s, 1H), 7.64 (dd, J=8.0, 41.6 Hz,1H), 7.51-7-47 (m, 4H), 7.38-7.36 (m, 2H), 7.29 (t, J=7.6 Hz, 4H), 7.20(t, J=7.2 Hz, 2H), 6.86 (dd, J=3.6, 8.8 Hz, 2H), 6.14 (dd, J=6.8, 106.8Hz, 1H), 5.69 (dd, J=8.0, 66.4 Hz, 1H), 3.82-3.63 (m, 7H), 3.61-3.42 (m,5H), 3.39-3.28 (m, 7H), 3.20-2.93 (m, 1H), 2.75-2.72 (m, 1H), 2.68-2.64(m, 1H), 1.10 (dd, J=4.8, 6.8 Hz, 6H), 1.00 (dd, J=6.4, 36.4 Hz, 6H).³¹P-NMR (162 MHz, DMSO-d₆): 148.37, 147.75. ¹⁹F-NMR (376 MHz, DMSO-d6):−73.87, −74.25, −74.62, −78.45, −78.83, −78.99, −79.37. ESI-LCMS: m/z808 [M−H]⁻.

Example 10

Preparation of Intermediate (14-17):

A solution of 14-14 (7.1 g, 9.9 mmol), DMAP (2.4 g, 19.9 mmol) and TEA(2.0 g, 19.9 mmol, 2.77 mL) in dry ACN (100 mL) was added TIPSCl (4.5 g,14.9 mmol). After the mixture was stirred at r.t. for 3 h, con.NH₄OH (30mL) was added, and the reaction was stirred at r.t. for 6 h. Upon ofcompletion, the solvent was removed, and the residue was dissolved inEA, washed with sat NH₄Cl (aq.) and brine, dried over anhydrous Na₂SO₄,concentrated to obtain a residue which was purified by columnchromatography to give 14-17 (6.1 g, 8.5 mmol, 86.03% yield) as a whitesolid. ESI-LCMS: m/z 713 [M+H]⁺.

Preparation of Intermediate (14-18):

To a solution of 14-17 (5.1 g, 7.1 mmol) in pyridine (50 mL) at 0° C.,was added BzCl (1.5 g, 10.7 mmol) dropwise. After the mixture wasstirred for 1 h at r.t., 200 mL H₂O and 300 mL EA were added to separatethe solution, the aqueous was extracted by EA, the combined organiclayer was washed with brine, dried over Na₂SO₄, concentrated to obtainthe crude product 14-18 (6.2 g) as a yellow oil. ESI-LCMS: m/z 817[M+H]⁺.

Preparation of Intermediate (14-19):

A solution of the crude product 14-18 (6.2 g) in pyridine (60 mL) wasadded 2N NaOH (in MeOH:H₂O=4:1) (30 mL) dropwise at 0° C., the mixturewas stirred at 0° C. for 15 min. Neutralized with saturated NH₄Cl (aq.)to give the pH=7˜8, and 300 mL H₂O and 400 mL EA were added in toseparate the solution, the aqueous was extracted by EA, the combinedorganic layer was washed with brine, dried over Na₂SO₄, concentrated toobtain the crude product which was purified by MPLC to give 14-19 (4.1g, 5.7 mmol, 80.3% yield over two steps) as a white solid. ¹H-NMR (400MHz, DMSO-d₆): δ=11.30 (s, 1H, exchanged with D₂O), 8.47 (d, J=7.6 Hz,1H), 8.02 (d, J=7.6 Hz, 2H), 7.63 (t, J=7.2 Hz, 1H), 7.54-7.45 (m, 6H),7.35-7.25 (m, 7H), 7.19 (t, J=7.2 Hz, 2H), 6.83 (d, J=8.8 Hz, 2H), 6.10(d, J=3.6 Hz, 1H), 5.19 (t, J=4.0 Hz, 1H), 3.77-3.70 (m, 6H), 3.64-3.61(m, 2H), 3.46-3.43 (m, 1H), 3.35 (s, 3H), 3.31-3.26 (m, 1H), 3.03 (d,J=8.8 Hz, 1H, exchanged with D₂O). ¹⁹F-NMR (376 MHz, DMSO-d₆): −73.47,−73.85, −78.26, −79.64. ESI-LCMS: m/z 713 [M+H]⁺.

Preparation of (14-20):

A solution of 14-19 (1.7 g, 2.3 mmol) and DCI (170 mg, 1.1 mmol) in dryDCM (20 mL) was added CEP[N(iPr)₂]₂ (790 mg, 2.6 mmol) under Ar. Themixture was stirred at r.t. for 1 h. The reaction was washed with 10%NaHCO₃(aq.) and brine, dried over Na₂SO₄ and concentrated to obtain thecrude product which was purified by column chromatography. This resultedin to give 14-20 (1.8 g, 1.9 mmol, 82.7% yield) as a white solid. ¹H-NMR(400 MHz, DMSO-d₆): δ=11.35 (s, 1H), 8.20 (dd, J=8.0, 81.6 Hz, 1H),8.03-8.00 (m, 2H), 7.64 (t, J=7.6 Hz, 1H), 7.54-7.44 (m, 6H), 7.37-7.16(m, 9H), 6.82 (dd, J=9.2, 16.4 Hz, 2H), 6.14 (dd, J=4.8, 90.4 Hz, 1H),4.15-4.02 (m, 1H), 3.90-3.40 (m, 11H), 3.36-3.24 (m, 6H), 3.12-2.98 (m,1H), 2.77-2.73 (m, 2H), 1.13 (d, J=6.8 Hz, 6H), 1.05 (dd, J=6.8, 28.0Hz, 6H). ³¹P-NMR (162 MHz, DMSO-d₆): 148.20, 147.96. ¹⁹F-NMR (376 MHz,DMSO-d₆): −73.36, −73.74, −73.98, −74.36, −77-89, −78.28, −78.55,−78.94. ESI-LCMS: m/z 913 [M+H]⁺.

Example 11

Preparation of Intermediate (15-21):

To a solution of compound 15-1 (90 g, 238.5 mmol) in dry Acetonitrile(900 mL) was added N-(5H-Purin-6-yl)benzamide (85.6 g, 357.7 mmol) andBSA (155.3 g, 763.2 mmol). The resulting suspension was stirred at 50°C. for 30 min. Then the mixture was at −5° C. and TMSOTf (79.5 g, 357.8mmol) was added by syringe. Then the mixture was stirred at 80° C. for72 h under N₂. The mixture was cooled and concentrated. And then addedwater and extracted with EA. The extract was washed with brine, driedover Na₂SO₄, and concentrated to give the residue which was purified bycolumn chromatography to afford 15-21 (120 g, 215.62 mmol, 90.4% yield)as a yellow solid. ¹H-NMR (400 MHz, DMSO-d₆): δ=8.56 (s, 1H), 8.53 (s,1H), 8.00 (t, J=8.0 Hz, 2H), 7.76-7.74 (d, J=8.0 Hz, 2H), 7.64-7.60 (t,J=7.5 Hz, 1H), 7.54-7.50 (t, J=7.68 Hz, 2H), 7.27-7.25 (d, J=7.8 Hz,2H,), 6.31 (d, J=3.3 Hz, 1H), 6.14-6.11 (m, 1H), 5.05-5.01 (t, J=8.0 Hz,1H), 4.67-4.63 (m, 1H), 4.53-4.49 (m, 1H), 4.37-4.34 (m, 1H), 2.31 (s,3H), 2.12 (s, 3H). ESI-LCMS: m/z 556 [M+H]⁺.

Preparation of Intermediate (15-22):

To a solution of 15-21 (120 g, 215.6 mmol) and Methylamine (267.9 g, 8.6mol) in Methanol (1300 mL) was stirred at r.t for 18 h under N₂. Themixture was concentrated and stirred at 25% EA in PET for 1 h to afford15-22 (52 g, 1778.0 mmol, 82.5% yield) as a white solid. ¹H-NMR (400MHz, DMSO-d₆): δ=8.36 (s, 1H), 8.15 (s, 1H), 7.40 (s, 2H), 6.24-6.22 (d,J=8.0 Hz, 1H), 5.91-5.90 (d, J=4 Hz, 1H), 5.63-5.60 (m, 1H), 5.04-5.00(m, 1H,), 4.34˜4.32 (m, 1H), 3.99-3.98 (d, J=4 Hz, 1H), 3.72-3.55 (m,2H). ESI-LCMS: m/z 292 [M+H]⁺.

Preparation of Intermediate (15-23):

To a solution of 15-22 (50 g, 171.1 mmol) and DMTrCl (121.7 g, 359.3mmol) in pyridine (250 mL) was stirred for 2 h at r.t. The mixture wasquenched with water, extracted with EA, dried over Na₂SO₄, concentratedto give the residue which was purified by column chromatography toafford 15-23 (117 g, 130.4 mmol, 76.2% yield) as a yellow solid. ¹H-NMR(400 MHz, DMSO-d₆): δ=8.29 (s, 1H), 7.76 (s, 1H), 7.20-7.12 (m, 19H),6.74 (m, 8H), 5.87 (s, 1H), 5.08 (s, 1H), 4.39 (s, 1H), 3.62 (s, 12H),3.32-3.10 (m, 2H). ESI-LCMS: m/z 896 [M+H]⁺.

Preparation of Intermediate (15-24):

To a solution of 15-23 (115 g, 128.2 mmol) and THF (800 mL) was stirredat 5° C. under N₂. NaH (4.0 g, 167.4 mmol) was slowly added below 10°C., and Sodium chlorodifluoroacetate (25.5 g, 167.4 mmol) and TBAI (5.6g, 16.7 mmol) were added at 5° C. The reaction was refluxed for 18 h.Cooled to r.t., the reaction was quenched with saturated NH₄Cl (aq.) andextracted with EA, washed with brine, dried over Na₂SO₄, concentrated togive the residue which was purified by column chromatography to give15-24 (56 g, 56.6 mmol, 48.3% yield) as a yellow crude solid. ESI-LCMS:m/z 990 [M+H]⁺.

Preparation of Intermediate (15-25):

To a solution of 15-24 (56 g, 57.3 mmol), methyl p-toluenesulfonate(15.8 g, 84.8 mmol) and K₂CO₃ (15.6 g, 113.0 mmol) in dry DMF (560 mL)was stirred at r.t. for 18 h under N₂. The mixture was quenched withwater, extracted with EA, wash with brine and concentrated to obtain aresidue which was purified by column chromatography to afford 15-25 (33g, 32.8 mmol, 58.1% yield) as a white solid. ¹H-NMR (400 MHz, DMSO-d₆):δ=8.40 (s, 1H), δ=7.82 (s, 1H), 7.40 (s, 1H), 7.31-7.16 (m, 18H),6.82-6.79 (m, 8H), 6.24-6.23 (d, J=4.0 Hz, 1H), 6.00-5.99 (t, 1H),4.88-4.85 (t, 1H), 4.17-4.14 (m, 1H), 3.79 (s, 3H), 3.68 (s, 12H),3.31-3.30 (m, 2H). ¹⁹F-NMR (376 MHz, DMSO-d₆): −75.75, −76.14, −76.89,−77.28. ESI-LCMS: m/z 1004 [M+H]⁺.

Preparation of Intermediate (15-26):

To a solution of 15-25 (30 g, 29.9 mmol) in THF (300 mL) was added NaBH₄(1.7 g, 44.8 mmol) and EtOH (100 mL) at 5° C. The mixture was allowed tostir at r.t. for 1.5 h, and quenched with sat NH₄Cl (aq.), extractedwith EA, washed with brine, dried over Na₂SO₄, concentrated to obtain aresidue which was purified by column chromatography to afford 15-26 (22g, 22.5 mmol, 75.4% yield) as a yellow solid. ¹H-NMR (400 MHz, DMSO-d₆):δ=8.35 (s, 1H), 7.79 (s, 1H), 7.26-7.13 (m, 21H), 6.77-6.75 (m, 9H),6.14-6.13 (d, J=4.0 Hz, 1H), 5.90 (t, 1H), 4.74˜4.72 (t, 1H), 4.11 (s,1H), 3.63 (s, 12H), 3.28˜3.25 (m, 2H). ¹⁹F-NMR (376 MHz, DMSO-d₆):−79.94, −80.31, −80.77, −81.15. ESI-LCMS: m/z 976 [M+H]⁺.

Preparation of Intermediate (15-27):

To a solution of 15-26 (20 g, 20.5 mmol) in THF (100 mL) were added NaH(491.7 mg, 20.5 mmol) and Methyl p-toluenesulfonate (11.4 g, 61.5 mmol)at 5° C. The mixture was allowed to stir at r.t. for 3 h, and was pouredinto cold water, extracted with EA, washed with brine, dried overNa₂SO₄, concentrated to obtain a residue which was purified by columnchromatography to afford 15-27 (16.5 g, 16.7 mmol, 81.3% yield) as ayellow solid. ¹H-NMR (400 MHz, DMSO-d₆): δ=8.40 (s, 1H), 7.86 (s, 1H),7.37-7.30 (m, 4H), 7.28-7.26 (m, 5H), 7.24-7.18 (m, 12H), 6.84-6.81 (m,8H), 6.17-6.16 (d, J=4.0 Hz, 1H), 5.92-5.89 (t, 1H), 4.79˜4.76 (t, 1H),4.15˜4.11 (m, 1H), 3.70 (s, 12H), 3.31 (s, 5H). ¹⁹F-NMR (376 MHz,DMSO-d₆): −76.83, −77.22, −78.05, −78.43. ESI-LCMS: m/z 990 [M+H]⁺.

Preparation of Intermediate (15-28):

To a solution of 15-27 (15.5 g, 15.6 mmol) in DCM (155 mL) was added asolution of TsOH (6.2 g, 36.0 mmol) in MeOH (30 mL). The mixture wasstirred at r.t. for 0.5 h, and washed with saturated NaHCO₃ to pH=7˜8,extracted with EA, dried over Na₂SO₄, concentrated to obtain a residuewhich was purified by column chromatography to give 15-28 (4.6 g, 11.4mmol, 72.8% yield) as a white solid. ESI-LCMS: m/z 386 [M+H]⁺.

Preparation of Intermediate (15-29):

To a solution of 15-28 (4.4 g, 11.3 mmol) in pyridine (40 mL) was slowlyadded BzCl (8.0 g, 56.7 mmol) at 5° C. The mixture was allowed to stirat r.t. for 2 h, and quenched with little water, ammonium hydroxide wasslowly added at 4° C. until intermediate product was disappeared. Themixture was extracted with EA, washed with brine, dried over Na₂SO₄,concentrated to obtain a crude product which was purified by columnchromatography. This resulted in to give 15-29 (6.0 g, 10.1 mmol, 89.1%yield) as a white solid. ¹H-NMR (400 MHz, DMSO-d₆): δ=8.67-8.66 (t, 1H),8.47-8.44 (t, 1H), 8.02-7.97 (m, 4H), 7.69-7.62 (m, 2H), 7.56-7.52 (m,4H), 6.32-6.31 (d, J=4.0 Hz, 1H), 6.09˜6.06 (m, 1H), 5.00˜4.98 (m, 1H),4.71-4.67 (m, 1H), 4.58˜4.54 (m, 1H), 4.43˜4.41 (m, 1H), 3.77˜3.72 (m,2H), 3.31 (m, 3H). ¹⁹F-NMR (376 MHz, DMSO-d₆): −76.97, −77.35, −78.19,−78.57. ESI-LCMS: m/z 594 [M+H]⁺.

Preparation of Intermediate (15-30):

To a solution of 15-29 (6.0 g, 10.1 mmol) in THF (70 mL) was added PPh₃(4.0 g, 15.1 mmol) and water (1.8 g, 100.9 mmol) under N₂. The mixturewas stirred at 45° C. for 15 h, poured into cold water, adjusted topH=1-2 by 2N HCl (aq.), extracted with EA. Then the aqueous phase wasadjusted to pH=7˜8 with saturated NaHCO₃(aq.), extracted with EA, washedwith brine, dried over Na₂SO₄, concentrated to afford 15-30 (5.5 g, 9.7mmol, 95.9% yield) as a white solid. ¹H-NMR (400 MHz, DMSO-d₆): δ=8.67(s, 2H), 8.03-8.01 (m, 2H), 7.88-7.86 (m, 2H), 7.65-7.61 (m, 2H),7.56-6.46 (m, 5H), 6.30˜6.29 (d, J=4.0 Hz, 1H), 5.42˜5.40 (m, 1H),4.69˜4.66 (m, 1H), 4.51˜4.47 (m, 1H), 4.08˜4.09 (m, 2H), 3.87-3.80 (m,2H), 3.46 (s, 5H). ¹⁹F-NMR (376 MHz, DMSO-d₆): −75.65, −76.03, −77.47,−77.86. ESI-LCMS: m/z 568 [M+H]⁺.

Preparation of Intermediate (15-31):

To a solution of 15-30 (5.5 g, 9.6 mmol) in dry DCM (55 mL) was addedMMTrCl (4.5 g, 14.5 mmol) and TEA (2.0 g, 19.3 mmol) under N₂. Themixture was allowed to stir at r.t. for 1.0 h, and quenched with water,extracted with DCM, washed with brine, dried over Na₂SO₄, concentratedto obtain a residue which was purified by column chromatography toafford 15-31 (7.3 g, 8.7 mmol, 89.8% yield) as a yellow solid. ¹H-NMR(400 MHz, DMSO-d₆): δ=8.46 (s, 1H), 8.41 (s, 1H), 8.00-7.98 (d, J=8.0Hz, 2H), 7.62-7.44 (m, 12H), 7.38-7.34 (m, 4H), 7.17-7.12 (m, 4H),7.06˜7.01 (m, 2H), 6.67˜6.65 (d, J=8.0 Hz, 2H), 6.17 (s, 1H), 4.57˜4.54(d, J=12 Hz, 1H), 4.35˜4.18 (m, 4H), 3.70˜3.65 (m, 2H), 3.50 (s, 3H),3.31 (s, 3H). ¹⁹F-NMR (376 MHz, DMSO-d₆): −73.61, −74.00, −77.94,−78.32. ESI-LCMS: m/z 840 [M+H]⁺.

Preparation of Intermediate (15-32):

To a solution of 15-31 (7.2 g, 8.5 mmol) in pyridine (70 mL) was slowlyadded 2N NaOH (aq.) (510.0 mg, 10.3 mmol) at 5˜10° C., The mixture wasallow to stir at r.t. for 10 min, neutralized with sat NH₄Cl (aq.) to pH7˜8, extracted with EA, washed with brine, dried over Na₂SO₄,concentrated to obtain a crude product which was purified by columnchromatography. This resulted in to give 15-32 (5.8 g, 7.9 mmol, 92.2%yield) as a white solid. ¹H-NMR (400 MHz, DMSO-d₆): δ=8.63 (s, 2H),7.99-7.97 (d, J=8.0 Hz, 2H), 7.64-7.60 (m, 1H), 7.54-7.50 (m, 2H),7.42-7.38 (m, 4H), 7.27-7.25 (d, J=8.0 Hz, 2H), 7.21-7.16 (m, 4H),7.12-7.09 (m, 2H), 6.72-6.70 (d, J=8.0 Hz, 2H), 6.28-6.27 (d, J=8.0 Hz,1H), 5.59 (m, 1H), 3.96 (s, 4H), 3.76-3.74 (m, 1H), 3.68-3.63 (m, 2H),3.60 (s, 3H), 3.47-3.41 (m, 2H), 3.27 (s, 3H). ¹⁹F-NMR (376 MHz,DMSO-d₆): −74.13, −74.51, −78.09, −78.47. ESI-LCMS: m/z 736 [M+H]⁺.

Preparation of (15-33):

To a solution of 15-32 (3 g, 4.1 mmol) and DCI (240.4 mg, 2.0 mmol) indry DCM (30 mL) was added CEP[N(iPr)₂]₂ (1.5 g, 4.9 mmol) under N₂. Themixture was allowed to stir at r.t. for 1 h, quenched with sat NaHCO₃,washed with brine, dried over Na₂SO₄, concentrated to obtain a crudeproduct which was purified by column chromatography. This resulted in togive 15-33 (3.1 g, 3.3 mmol, 81.2% yield) as a white solid. ¹H-NMR (400MHz, DMSO-d₆): δ=11.20 (s, 1H), 8.69-8.68 (d, J=4.0 Hz, 1H), 8.50-8.48(d, J=8.0 Hz, 1H), 8.05-8.03 (m, 2H), 7.66-7.62 (m, 1H), 7.57-7.45 (m,6H), 7.40-7.33 (m, 2H), 7.30-7.11 (m, 6H), 6.85-6.76 (m, 2H), 6.45-6.30(m, 1H), 4.77-4.74 (m, 0.5H), 4.00-3.99 (m, 0.5H), 3.86-3.84 (m, 0.5H),3.77-3.70 (m, 5H), 3.67-3.59 (m, 4H), 3.56-3.51 (m, 1H), 3.50-3.34 (m,3H), 3.32 (s, 3H), 3.08-3.06 (m, 0.5H), 2.77-2.65 (m, 2H), 1.08-0.99 (m,12H). ¹⁹F-NMR (376 MHz, DMSO-d6): −73.92, −74.27, −74.30, −74.65,−77.64, −78.03, −78.55, 78.93. ESI-LCMS: m/z 936 [M+H]⁺.

Example 12

Preparation of Intermediate (16-34):

A solution of 16-1 (150.0 g, 397.5 mmol) and 6-Chloroguanine (101.1 g,596.3 mmol) in dry ACN (1.5 L) was added BSA (259.5 g, 1.27 mol) underAr. The resulting suspension was stirred at 50° C. for 1 h. Then themixture was cooled at −5° C. and TMSOTf (590.5 g, 2.66 mol) was added bysyringe. After that the mixture was stirred at 60° C. for 5 h,concentrated to remove ACN and the residue was dissolved in EA (2.0 L),washed with sat NaHCO₃(aq.) and brine, dried over Na₂SO₄, concentratedto give the crude product 16-34 (265.0 g) as a yellow solid. ESI-LCMS:m/z 487 [M+H]⁺.

Preparation of Intermediate (16-35):

A solution of 16-34 (crude, 265 g, 353.8 mmol) and TEA (107.4 g, 1.06mol, 148 mL) in DCM (2.5 L) was added MMTrCl (164.0 g, 530.7 mmol) andDMAP (8.6 g, 70.8 mmol), the mixture was stirred at r.t. for 5 h, washedwith water and brine, dried over Na₂SO₄, concentrated to obtain theresidue which was purified by column chromatography to give 16-35 (160.0g, 210.8 mmol, 53.0% yield over 2 steps) as a yellow solid. ESI-LCMS:m/z 759 [M+H]⁺.

Preparation of Intermediate (16-36):

To a solution of 35 (20.0 g, 26.39 mmol) in Benzyl alcohol (200 mL) wasadded NaH (5.3 g, 131.93 mmol) at 0° C. The resulting mixture wasallowed to stir at r.t. for 2 h, quenched with cold water, extractedwith EA, washed with water and brine, dried over Na₂SO₄, concentrated toobtain a residue which was purified by column chromatography to give16-36 (11.2 g, 16.72 mmol, 63.1% yield) as white solid. ESI-LCMS: m/z671 [M+H]⁺.

Preparation of Intermediate (16-37):

A solution of 16-36 (49.0 g, 73.1 mmol) and pyridine (28.9 g, 365.3mmol, 29 mL) in DCM (500 mL) was added DMTrCl (27.2 g, 80.4 mmol) atr.t. The mixture was stirred at r.t. for 3 h, washed with water andbrine, dried over Na₂SO₄, concentrated to obtained a residue which waspurified by column chromatography to give 16-37 (58.9 g, 60.6 mmol,82.9% yield) as a white solid. ESI-LCMS: m/z 973 [M+H]⁺.

Preparation of Intermediate (16-38):

A solution of 16-37 (58.9 g, 60.6 mmol) in dry THF (300 mL) were addedNaH (5.1 g, 127.8 mmol, 60% purity) and Methyl p-toluenesulfonate (19.5g, 127.8 mmol) at 0° C., the mixture was allowed to stirred at r.t., andTBAI (4.7 g, 12.8 mmol) was added. After that the mixture was stirred at80° C. for 24 h, and quenched with sat NH₄Cl(aq.), extracted with EA,washed with brine, dried over Na₂SO₄, concentrated to obtain a residuewhich was purified by column chromatography to give the crude product16-38 (30.9 g) as a blank solid. ESI-LCMS: m/z 1067 [M+H]⁺.

Preparation of Intermediate (16-39):

A solution of the crude 16-38 (30.9 g) and K₂CO₃ (8.0 g, 58.1 mmol) inDMF (200 mL) was added Sodium chlorodifluoroacetate (5.4 g, 29.1 mmol)at r.t. The mixture was stirred at r.t. for 15 h, quenched with satNH₄Cl(aq.), extracted with EA, washed with brine, dried over Na₂SO₄,concentrated to obtain a residue which was purified by columnchromatography to give 16-39 (9.8 g, 9.1 mmol, 15.0% yield over 2 steps)as a yellow solid. ESI-LCMS: m/z 1081 [M+H]⁺.

Preparation of Intermediate (16-40):

A solution of 16-39 (9.3 g, 8.6 mmol) in THF (180 mL) was added NaBH₄(488 mg, 12.9 mmol) and EtOH (15 mL) at 0° C. The mixture was stirred at0° C. for 1 h, quenched with sat NH₄Cl (aq.), extracted with EA, washedwith brine, dried over Na₂SO₄, concentrated to obtain a residue whichwas purified by column chromatography. This resulted in to give 16-40(8.5 g, 7.0 mmol, 81.63% yield, 87% purity) as a white solid. ESI-LCMS:m/z 1053 [M+H]⁺.

Preparation of Intermediate (16-41):

A solution of 16-40 (8.3 g, 7.9 mmol) in THF (80 mL) was added NaH (472mg, 11.8 mmol, 60% purity) and Methyl p-toluenesulfonate (2.9 g, 15.8mmol) at 0° C. The mixture was allowed to stir at r.t. for 15 h, andpoured into cold water, extracted with EA, washed with brine, dried overNa₂SO₄, concentrated to obtained the crude product 16-41 (9.1 g) as ayellow oil. ESI-LCMS: m/z 1067 [M+H]⁺.

Preparation of Intermediate (16-42):

A solution of crude 16-41 (9.1 g) in DCM (100 mL) was added a solutionof p-TsOH (10 g) in MeOH (10 mL). The mixture was stirred at r.t. for0.5 h, and neutralized with TEA to give pH=7, concentrated to obtain aresidue which was purified by column chromatography. This resulted in togive 16-42 (3.2 g, 6.5 mmol, 82.3% yield over 2 steps) as a yellowsolid. ¹H-NMR (400 MHz, DMSO-d₆): δ=8.14 (s, 1H), 7.52-7.50 (m, 2H),7.42-7.33 (m, 3H), 6.62 (s, 2H, exchanged with D₂O), 6.01 (d, J=6.4 Hz,1H), 5.62 (t, J=6.0 Hz, 1H), 5.49 (t, J=12.8 Hz, 2H), 5.39 (t, J=5.6 Hz,1H, exchanged with D₂O), 4.64 (dd, J=3.6, 5.6 Hz, 1H), 4.02 (dd, J=3.6,7.2 Hz, 1H), 3.74-3.66 (m, 3H), 3.64-3.59 (m, 1H), 3.32 (s, 3H). ¹⁹F-NMR(376 MHz, DMSO-d₆): −77.52, −77.91, −78.17, −78.55). ESI-LCMS: m/z 493[M+H]⁺.

Preparation of Intermediate (16-43):

A solution of 16-42 (3.2 g, 6.5 mmol) in pyridine (30 mL) was addedi-BuCl (2.1 g, 19.5 mmol) drop wise at 0° C. The mixture was stirred at0° C. for 1 h, quenched with MeOH and concentrated to obtain a residuewhich was purified by column chromatography. This resulted in to give16-43 (3.2 g, 5.5 mmol, 84.59% yield) as a yellow oil. ¹H-NMR (400 MHz,DMSO-d₆): δ=10.60 (s, 1H, exchanged with D₂O), 8.42 (s, 1H), 7.57-7.54(m, 2H), 7.44-7.35 (m, 3H), 6.20 (d, J=2.8 Hz, 1H), 5.71 (dd, J=2.4, 5.6Hz, 1H), 5.68-5.64 (m, 3H), 4.41 (dd, J=3.6, 12.0 Hz, 1H), 4.24 (dd,J=5.6, 12.0 Hz, 1H), 4.19-4.15 (m, 1H), 3.80 (t, J=10.4 Hz, 2H), 3.38(s, 3H), 3.35 (s, 3H), 2.45-2.38 (m, 1H), 1.12 (dd, J=2.4, 6.8 Hz, 6H),0.97 (dd, J=6.8, 16.4 Hz, 6H). ¹⁹F-NMR (376 MHz, DMSO-d₆): −76.48,−76.87, −77.62, −78.01). ESI-LCMS: m/z 633 [M+H]⁺.

Preparation of Intermediate (16-44):

A solution of 16-43 (1.6 g, 2.5 mmol) in THF (20 mL) was added Pd/C (200mg), the mixture was stirred at r.t. for 5 h under H₂. The reaction wasfiltered and the filtrate was concentrated to give 16-44 (1.2 g, 2.3mmol, 91.86% yield) as a white solid. ¹H-NMR (400 MHz, DMSO-d₆): δ=11.60(s, 1H, exchanged with D₂O), 8.22 (s, 1H), 6.05 (d, J=2.8 Hz, 1H), 5.14(dd, J=3.2, 5.6 Hz, 1H), 4.40 (dd, J=2.4, 12.0 Hz, 1H), 4.17 (dd, J=5.6,12.0 Hz, 1H), 3.93-3.73 (m, 4H), 3.38 (s, 3H), 1.14 (d, J=7.2 Hz, 6H),1.04 (dd, J=7.2, 12.4 Hz, 6H). ¹⁹F-NMR (376 MHz, DMSO-d₆): −75.73,−76.12, −77.46, −77.84). ESI-LCMS: m/z 517 [M+H]⁺.

Preparation of Intermediate (16-45):

A solution of 16-44 (3.0 g, 5.8 mmol) and collidine (1.8 g, 14.5 mmol)in DCM (40 mL) was added MMTrCl (2.7 g, 8.7 mmol) and AgNO₃ (986 mg, 5.8mmol), the mixture was stirred at r.t. for 1 h, filtered and thefiltrate was washed with water and dried over anhydrous Na₂SO₄,concentrated to obtain a residue which was purified by columnchromatography to give 16-45 (3.8 g, 4.8 mmol, 82.94% yield) as a whitesolid. ¹H-NMR (400 MHz, DMSO-d₆): δ=12.16 (s, 1H, exchanged with D₂O),11.48 (s, 1H, exchanged with D₂O), 7.86 (s, 1H), 7.50 (dd, J=5.2, 7.2Hz, 4H), 7.37 (d, J=8.8 Hz, 2H), 7.28 (t, J=7.2 Hz, 4H), 7.20 (t, J=7.2Hz, 2H), 6.83 (d, J=9.2 Hz, 2H), 6.11 (d, J=3.2 Hz, 1H), 4.18 (dd,J=5.2, 8.8 Hz, 1H), 4.08 (t, J=4.4 Hz, 1H), 4.44-3.97 (m, 1H), 3.74 (dd,J=7.6, 10.4 Hz, 2H), 3.70 (s, 3H), 3.46-3.41 (m, 1H), 3.16 (d, J=8.0 Hz,1H, exchanged with D₂O), 2.86-2.79 (m, 1H), 2.33-2.26 (m, 1H), 1.13 (t,J=7.2 Hz, 6H), 0.89 (dd, J=7.2, 14.8 Hz, 6H). ¹⁹F-NMR (376 MHz,DMSO-d₆): −74.01, −74.39, −78.00, −78.38). ESI-LCMS: m/z 789 [M+H]⁺.

Preparation of Intermediate (16-46):

A solution of 16-45 (3.8 g, 4.8 mmol) in pyridine (40 mL) was added 2NNaOH (MeOH:H₂O=4:1) (15 mL) at 0° C., the mixture was stirred at 0° C.for 1 h. Then the reaction was neutralized with saturated NH₄Cl (aq) topH=7˜8, extracted with EA, washed with brine, dried over anhydrousNa₂SO₄, concentrated to obtain a residue which was purified by columnchromatography. This resulted in to give 16-46 (3.0 g, 4.2 mmol, 87.50%yield) as a white solid. ¹H-NMR (400 MHz, DMSO-d₆): δ=12.16 (s, 1H,exchanged with D₂O), 11.68 (s, 1H, exchanged with D₂O), 8.19 (s, 1H),7.51 (dd, J=5.2, 7.2 Hz, 4H), 7.38 (d, J=8.8 Hz, 2H), 7.31 (t, J=7.2 Hz,4H), 7.21 (t, J=7.2 Hz, 2H), 6.87 (d, J=8.8 Hz, 2H), 6.26 (d, J=6.0 Hz,1H), 5.15 (t, J=4.4 Hz, 1H), 4.76 (t, J=6.0 Hz, 1H, exchanged with D₂O),3.75-3.70 (m, 5H), 3.42 (d, J=2.4 Hz, 1H), 3.38-3.28 (m, 5H), 3.23 (d,J=6.4 Hz, 1H, exchanged with D₂O), 3.16-3.10 (m, 1H), 2.88-2.81 (m, 1H),1.15 (dd, J=6.4, 7.2 Hz, 6H). ¹⁹F-NMR (376 MHz, DMSO-d₆): −74.73,−75.12, −78.79, −79.17. ESI-LCMS: m/z 719 [M+H]⁺.

Preparation of (16-47): A solution of 16-46 (3.0 g, 4.2 mmol) and DCI(394 mg, 3.3 mmol) in dry DCM (50 mL) was added CEP[N(iPr)₂]₂ (1.5 g,5.0 mmol) under Ar. The mixture was stirred at r.t. for 1 h. Thereaction was washed with 10% NaHCO₃(aq) and water, dried over Na₂SO₄ andconcentrated to obtain the crude product which was purified by columnchromatography. This resulted in to give 16-47 (3.0 g, 3.2 mmol, 77.81%yield) as a white solid. H-NMR (400 MHz, DMSO-d₆): δ=12.13 (s, 1H),11.65 (d, J=5.6 Hz, 1H), 8.11 (s, 0.35H), 7.95 (s, 0.65H), 7.50 (t,J=7.2 Hz, 4H), 7.38-7.26 (m, 6H), 7.20 (dd, J=8.4, 16.0 Hz, 2H), 6.88(d, J=9.2 Hz, 0.7H), 6.82 (d, J=8.8 Hz, 1.3H), 6.31 (d, J=6.8 Hz,0.35H), 6.14 (d, J=4.4 Hz, 0.65H), 4.97 (t, J=6.4 Hz, 0.35H), 3.97 (t,J=4.4 Hz, 0.65H), 3.82-3.54 (m, 9H), 3.48-3.14 (m, 9H), 2.86-2.69 (m,2H), 1.24-0.92 (m, 18H). ³¹P-NMR (162 MHz, DMSO-d₆): 148.35, 148.24.ESI-LCMS: m/z 919 [M+H]⁺.

Example 13

Preparation of Intermediate (17-2):

To a solution of 17-1 (120 g, 318 mmol) in ACN (1.2 L) was added BSA(208 g, 1016 mmol) and N⁴-Benzoylcytosine (102 g, 474 mmol). The mixturewas stirred at 50° C. for 1 h until clear. Then the solution was cooledto −10° C. TMSOTf (84 g, 381 mmol) was slowly added to the mixture. Thenwarmed up. The reaction mixture was stirred at 60° C. for 12 h. Themixture was cooled to 0° C. and saturated NaHCO₃ was added until pH=7.5.The mixture was filtered and the filtrate was concentrated to give theresidue. The residue was dissolved in EA and washed with water andbrine, dried over Na₂SO₄. The organic solution was concentrated to givethe crude 17-2 (160 g) as a yellow oil. ESI-LCMS. m/z 533 [M++H]⁺.

Preparation of Intermediate (17-3):

A solution of crude 17-2 (160 g) in Pyridine (1.5 L) was added to 600 mL2N NaOH solution in Methanol and Water at 0° C. The suspension wasstirred at 0° C. for 30 min. The reaction mixture was quenched byaddition of sat. NH₄Cl solution (1 L). The solution was added to water(15 L) to give the solid. The solid was filtered and washed with(PE:EA=3:1) to give 17-3 (80 g, 214 mmol, 67% over two steps) as whitesolid. ESI-LCMS: m/z 373 [M±H]⁺.

Preparation of Intermediate (17-4):

To a solution of 17-3 (80 g, 214 mmol) in Pyridine (1 L) was addedDMTrCl (87 g, 257 mmol). The reaction mixture was stirred at r.t. for 1h. MeOH (50 ml) was added to the mixture. The solution was concentratedto give the crude. The crude was dissolved in EA and washed with waterand brine. The organic layer was dried over Na₂SO₄. The organic solutionwas concentrated to give the crude 17-4 (160 g) as a yellow oil whichwas used directly for the next step. ESI-LCMS: m/z 675 [M+H]⁺.

Preparation of Intermediate (17-5):

To a solution of crude 17-4 (160 g) in DCM (1 L) was added Imidazole (22g, 332 mmol) and TBSCl (46 g, 310.62 mmol). The reaction mixture wasstirred at r.t. for 12 h. The mixture was diluted with DCM (1 L). Waterwas added. The organic layer was washed with brine and concentrated togive the crude 17-5 (200 g) which was used directly for the next step.ESI-LCMS: m/z 789 [M+H]⁺.

Preparation of Intermediate 17-6):

A solution of crude 17-5 (200 g) in Methylamine (1 L) was stirred atr.t. for 2 h. The solution was concentrated and purified by columnchromatography to give 17-6 (130 g, 204.42 mmol, 94.8% yield) as a whitesolid. ESI-LCMS: m/z 685 [M±H].

Preparation of Intermediate (17-7):

To a solution of 17-6 (142 g, 207.34 mmol) in DCM (1 L) was addedcollidine (50.25 g, 414.69 mmol) and MMTrCl (95.79 g, 311.01 mmol). ThenAgNO₃ (52.83 g, 311.01 mmol) was added to the solution. The reactionmixture was stirred at r.t. for 1 h. The mixture was filtered. Theorganic solution was washed with water. The organic layer wasconcentrated to give the crude 17-7 (190 g, 198.50 mmol, 95.73% yield)as a yellow solid which was used directly for the next step. ESI-LCMS:m/z 957.4 [M+H]⁺.

Preparation of Intermediate (17-8):

To a solution of 17-7 (190 g, 198.50 mmol) in THF (1 L) was added TBAF(77.85 g, 297.74 mmol). The mixture was stirred at r.t. for 12 h. Waterwas added. The product was extracted with EA. The organic layer waswashed with brine and dried over Na₂SO₄. The organic solution wasconcentrated and purified by column chromatography to give 17-8 (160 g,189.81 mmol, 95.6% yield) as a white solid. ESI-LCMS: m/z 843.4 [M+H]⁺.

Preparation of Intermediate (17-9):

To a solution of 17-8 (154 g, 182.70 mmol) in DMF (1 L) was added Ag₂O(84.68 g, 365.39 mmol, 11.86 mL),(2-Bromoethoxy)-tert-butyldimethylsilane (78.67 g, 328.85 mmol) and NaI(41.08 g, 274.04 mmol). The reaction mixture was stirred at 45° C. for12 h. Water was added. The mixture was filtered and product wasextracted with EA. The organic solution was concentrated and purified bycolumn chromatography to give 17-9 (90 g, 89.89 mmol, 49.2% yield) as ablack solid. ESI-LCMS: m/z 1001.4 [M+H]⁺.

Preparation of Intermediate (17-10):

To a solution of 17-9 (90 g, 89.89 mmol) in THF (500 mL) was added TBAF(35.25 g, 134.83 mmol). The reaction mixture was stirred at r.t. for 12h. Water was added to the mixture. The product was extracted with EA.The organic layer was concentrated and purified by column chromatographyto give 17-10 (75 g, 84.56 mmol, 94.1% yield) as a yellows solid.ESI-LCMS: m/z 887.2 [M+H]⁺.

Preparation of Intermediate (17-11):

To a solution of 17-10 (54 g, 60.88 mmol) in THF (400 mL) was added NaH(1.75 g, 73.06 mmol) at 0° C. After stirred at r.t. for 30 min, thereaction was cooled to 0° C., CS₂ (9.50 g, 121.76 mmol) was added to themixture. After 10 min at this temperature, MeI (15.45 g, 109.58 mmol)was added to the mixture. And then the mixture was stirred at r.t. for12 h. Water was added. The product was extracted with EA. The organiclayer was washed with brine and dried over Na₂SO₄. The organic solutionwas concentrated and purified by column chromatography to obtain 17-11(50 g, 51.17 mmol, 84.1% yield) as a white solid. ESI-LCMS: m/z 977.2[M+H]^(F)

Preparation of Intermediate (17-12):

To a solution of 17-11 (17 g, 17.40 mmol) in DCM (100 mL) was added PTSA(5.99 g, 34.79 mmol) in Methanol (10 mL). The mixture was stirred atr.t. for 1 h. Cooled to 0° C. Con. NH₄OH was added to give pH=7.5. Waterwas added and the product was extracted with DCM. The organic layer wasconcentrated and purified by column chromatography to give 17-12 (10 g,14.82 mmol, 85.2% yield) as a white solid. ESI-LCMS: m/z 675.1 [M+H]⁺.

Preparation of Intermediate (17-13):

To a solution of 17-12 (40 g, 59.28 mmol) in DCM (800 mL) was added TFA(150 mL). The mixture was stirred at r.t. for 1 h. Cooled to 0° C. Con.NH4OH was added to the solution to give pH=7.5. Water was added and theproduct was extracted with DCM. The organic layer solution was driedover Na₂SO₄ and concentrated to give the crude product which waspurified by column chromatography to give 17-13 (21 g, 52.18 mmol, 88.0%yield) as a white solid. ESI-LCMS: m/z 403.0 [M+H]⁺.

Preparation of Intermediate (17-14):

To a solution of 17-13 (26 g, 64.60 mmol) in Pyridine (500 mL) was addedAc₂O (19.79 g, 193.81 mmol). The reaction mixture was stirred at 40° C.for 1 h. Water was added. The solution was concentrated to give thecrude. The crude was purified by column chromatography and MPLC to give17-14 (21 g, 43.16 mmol, 66.8% yield) as a white solid. ESI-LCMS: m/z487.0 [M+H]⁻.

Preparation of Intermediate (17-15):

To a solution of DBH (4.19 g, 14.64 mmol) in DCM (75 mL) was addedHF/Pyridine (10 mL) slowly at −60° C. Then SM-1 (2.5 g, 5.14 mmol)dissolved in DCM was slowly added to the mixture. The reaction mixturewas stirred at −40° C. for 1 h. DCM was added to the mixture. Sat.NaHCO₃ was slowly added to the mixture to give the pH=7.0. The solutionwas concentrated to give the crude product. The crude was purified bycolumn chromatography and MPLC to give 17-15 (1.1 g, 2.37 mmol, 46.1%yield) as a white solid. ESI-LCMS: m/z 465.1 [M+H]⁺. ¹⁹F NMR (376 MHz,Chloroform-d) 6-55.49.

Preparation of Intermediate (17-16):

A solution of 17-15 (4.6 g, 9.91 mmol) in Methylamine (30 mL) wasstirred at r.t. for 1 h. The solution was concentrated and purified bycolumn chromatography to 17-16 (3.7 g, 9.73 mmol, 98.2% yield) as awhite solid. ESI-LCMS: m/z 381.1 [M+H]⁺.

Preparation of Intermediate (17-17):

To a solution of 17-16 (1.7 g, 4.47 mmol) in AcOH (5 mL) and Water (5mL) was added NaNO₂ (30.40 g, 447.04 mmol). The mixture was stirred atr.t. for 3 h. LC-MS showed 70% conversion. EA and water was added. Theorganic layer was concentrated and purified by MPLC to give 17-17 (1.5g, 3.93 mmol, 88.0% yield) as a white solid. ESI-LCMS: m/z 382.2 [M+H]⁺.

Preparation of Intermediate (17-18):

To a solution of 17-17 (2.23 g, 5.85 mmol) in DCM (30 mL) was addedImidazole (796.36 mg, 11.70 mmol) and TBSCl (1.32 g, 8.77 mmol). Thereaction mixture was stirred at r.t. for 12 h. Water was added. Theorganic layer was washed with brine and dried over Na₂SO₄. The organiclayer was concentrated to give the crude 17-18 (2.8 g, 5.65 mmol, 96.6%yield) as a white solid. ESI-LCMS: m/z 496.4 [M±H]⁺.

Preparation of Intermediate (17-19):

To a solution of 17-18 (2.8 g, 5.65 mmol) in THF (30 mL) was added PPh₃(1.78 g, 6.78 mmol) and water (203.59 mg, 11.30 mmol). The mixture wasstirred at 50° C. for 2 h under N₂ atmosphere. The mixture wasconcentrated and purified by column chromatography to give 17-19 (2.5 g,5.32 mmol, 94.2% yield) as a white solid. ESI-LCMS: m/z 470.3 [M+H]⁺.

Preparation of Intermediate (17-20):

To a solution of 17-19 (2.8 g, 5.96 mmol) in DCM (40 mL) was added TEA(1.21 g, 11.93 mmol, 1.66 mL) and MMTrCl (2.76 g, 8.95 mmol). Themixture was stirred at r.t. for 1 h. Water was added. The organic layerwas dried over Na₂SO₄ and concentrated, the residue was purified bycolumn chromatography to give 17-20 (3.1 g, 4.18 mmol, 70.1% yield) as awhite solid. ¹H NMR (400 MHz, DMSO-d₆) δ 11.36 (d, J=2.2 Hz, 1H), 7.67(d, J=8.0 Hz, 1H), 7.46 (td, J=8.6, 1.3 Hz, 4H), 7.37-7.30 (m, 2H), 7.25(t, J=7.5 Hz, 4H), 7.22-7.14 (m, 2H), 6.84-6.77 (m, 2H), 5.54 (s, 1H),5.44 (dd, J=8.0, 2.2 Hz, 1H), 4.17-4.01 (m, 4H), 3.90 (dd, J=9.8, 2.4Hz, 1H), 3.70 (s, 4H), 3.07 (td, J=10.1, 4.5 Hz, 1H), 3.03-2.93 (m, 1H),2.82 (d, J=10.3 Hz, 1H), 1.51 (d, J=4.5 Hz, 1H), 0.84 (s, 9H), 0.12 (s,3H), −0.03 (s, 3H). ¹⁹F NMR (376 MHz, DMSO-d₆) δ −58.72.

Preparation of Intermediate (17-21):

To a solution of 17-20 (3.1 g, 4.18 mmol) in THF (30 mL) was added TBAF(1.64 g, 6.27 mmol). The mixture was stirred at r.t. for 15 h. Themixture was concentrated and purified by column chromatography to give17-21 (2.2 g, 3.51 mmol, 83.9% yield) as a white solid. ¹H NMR (400 MHz,DMSO-d₆) δ 11.30 (d, J=2.0 Hz, 1H), 7.98 (d, J=8.1 Hz, 1H), 7.52-7.42(m, 4H), 7.37-7.30 (m, 2H), 7.30-7.23 (m, 4H), 7.23-7.14 (m, 2H),6.87-6.79 (m, 2H), 5.52 (d, J=2.0 Hz, 1H), 5.50 (d, J=1.5 Hz, 1H), 5.17(t, J=3.8 Hz, 1H), 4.13 (t, J=4.2 Hz, 2H), 4.05 (dd, J=4.2, 2.1 Hz, 2H),3.90 (dt, J=10.0, 2.2 Hz, 1H), 3.72 (s, 3H), 3.67 (dt, J=11.4, 3.7 Hz,1H), 3.39 (td, J=6.4, 5.1 Hz, 1H), 3.10 (td, J=10.3, 4.3 Hz, 1H), 2.97(dt, J=11.8, 4.7 Hz, 1H), 2.68 (d, J=10.6 Hz, 1H), 1.44 (d, J=4.3 Hz,1H). ¹⁹F NMR (376 MHz, DMSO-d₆) δ −58.82.

Preparation of (17-22):

To a solution of 17-21 (1.4 g, 2.23 mmol) in DCM (10 mL) was added DCI(318.88 mg, 2.23 mmol) and CEP[N(iPr)₂]₂ (872.87 mg, 2.90 mmol). Themixture was stirred at r.t. for 1 h. The solution was diluted with DCM.The organic layer was washed with sat. NaHCO₃, water and brine. Theorganic layer was dried over Na₂SO₄ and purified by columnchromatography to give 1.3 g crude product. The product was drying invacuum at 40° C. overnight. This resulted in (1.15 g, 1.39 mmol, 62.28%yield) to give 17-22 as a white solid. MS m/z [M−H]⁻ (ESI): 826.4; ¹HNMR (400 MHz, DMSO-d₆) δ 11.37 (s, 1H), 7.78 (d, J=8.1 Hz, 0.6 H),7.59-7.44 (m, 4.4 H), 7.41-7.33 (m, 2H), 7.27 (m, 4H), 7.24-7.14 (m,2H), 6.88-6.79 (m, 2H), 5.57-5.48 (m, 1.4H), 5.44 (d, J=8.0 Hz, 0.6H),4.25-4.13 (m, 2H), 4.13-3.90 (m, 3H), 3.79-3.38 (m, 8H), 3.03 (m, 2H),2.86 (dd, J=26.4, 10.4 Hz, 1H), 2.79-2.67 (m, 2H), 1.67 (d, J=4.5 Hz,0.4 H), 1.26 (d, J=4.5 Hz, 0.6 H), 1.18 (d, J=6.7 Hz, 3H), 1.15-1.02 (m,9H). ³¹P NMR (162 MHz, DMSO-d6) δ 148.03, 146.68.

Example 14

Preparation of Intermediate (18-23):

To a solution of 18-16 (1.2 g, 3.16 mmol) in Pyridine (10 mL) was addedBzCl (971.92 mg, 6.94 mmol) at 0° C. The mixture was stirred at r.t. for1 h. Water was added. The product was extracted with EA. The organiclayer was washed with brine and dried over Na₂SO₄. The organic layer wasconcentrated and purified by column chromatography to give 18-23 (1.58g, 2.68 mmol, 85.1% yield) as a white solid. ESI-LCMS: m/z 589.1 [M±H]⁺.

Preparation of Intermediate (18-24):

To a solution of 18-23 (1.47 g, 2.50 mmol) in THF (20 mL) was addedwater (90.00 mg, 5.00 mmol) and PPh₃ (786.20 mg, 3.00 mmol). The mixturewas stirred at 50° C. under N₂ atmosphere. The mixture was concentratedand purified by column chromatography to give 18-24 (1.35 g, 2.40 mmol,96.1% yield) as a white solid. ESI-LCMS: m/z 563.2 [M+H]⁺.

Preparation of Intermediate (18-25):

To a solution of 18-24 (1.4 g, 2.49 mmol) in DCM (40 mL) was added TEA(503.71 mg, 4.98 mmol, 694.29 uL) and MMTrCl (1.15 g, 3.73 mmol). Themixture was stirred at r.t. for 1 h. Water was added. The organic layerwas dried over Na₂SO₄ and concentrated, the residue was purified bycolumn chromatography to give 18-25 (2 g, 2.40 mmol, 96.3% yield) as awhite solid. ¹⁹F NMR (376 MHz, DMSO-d6) δ −58.72.

Preparation of Intermediate (18-26):

To a solution of 18-25 (2 g, 2.40 mmol) in Pyridine (30 mL) was addedNaOH (287.46 mg, 7.19 mmol) in Methanol (12 mL) and Water (3 mL) at 0°C. The mixture was stirred at r.t. for 0.5 h. Sat. NH₄Cl was added togive pH=8. The product was extracted with EA. The organic layer wasconcentrated and purified by MPLC to give 18-26 (1.5 g, 2.05 mmol,85.69% yield) as a white solid. ¹H NMR (400 MHz, DMSO-d₆) δ 11.27 (s,1H), 8.55 (d, J=7.5 Hz, 1H), 8.11-7.94 (m, 2H), 7.77-7.57 (m, 1H),7.57-7.40 (m, 6H), 7.40-7.10 (m, 9H), 6.97-6.76 (m, 2H), 5.56 (s, 1H),5.36-5.18 (m, 1H), 4.15 (dd, J=9.7, 5.4 Hz, 4H), 4.01 (d, J=10.2 Hz,1H), 3.84 (d, J=11.7 Hz, 1H), 3.72 (s, 3H), 3.18-2.97 (m, 2H), 2.66 (d,J=10.9 Hz, 1H), 1.48 (d, J=4.1 Hz, 1H). ¹⁹F NMR (376 MHz, DMSO-d₆) δ−58.75.

Preparation of (18-27):

To a solution of 18-26 (1.4 g, 1.92 mmol) in DCM (30 mL) was added DCI(226.08 mg, 1.92 mmol) and CEP[N(iPr)₂]₂ (749.69 mg, 2.49 mmol). Themixture was stirred at r.t. for 1 h. The solution was diluted with DCM.The organic layer was washed with water and brine. The organic layer wasdried over Na₂SO₄ and concentrated to the crude product. The crude waspurified by HPLC. The product was drying in vacuum at 40° C. overnight.This resulted in (1.65 g, 1.77 mmol, 92.51% yield) to give 18-27 as awhite solid. ESI-LCMS: m/z 877 [M+H]⁺. ¹H NMR (400 MHz, DMSO-d₆) δ 11.33(s, 1H), 8.34 (d, J=7.4 Hz, 0.6H), 8.20 (d, J=7.4 Hz, 0.6H), 8.02 (dt,J=8.3, 1.2 Hz, 2H), 7.73-7.57 (m, 1H), 7.57-7.40 (m, 6H), 7.40-7.30 (m,3H), 7.30-7.10 (m, 6H), 6.86-6.71 (m, 2H), 5.60 (d, J=1.1 Hz, 1H),4.41-4.03 (m, 5H), 4.00-3.39 (m, 8H), 3.05 (m, 2H), 2.92-2.70 (m, 3H),1.53 (d, J=4.2 Hz, 0.4H), 1.29 (d, J=4.2 Hz, 0.6H), 1.26-1.00 (m, 12H).³¹P NMR (162 MHz, DMSO-d₆) δ 148.41, 146.88.

Example 15

Preparation of Intermediate (19-2):

To a solution of 19-1 (48.0 g, 138.2 mmol) in anhydrous pyridine (500mL) was added TIPDSCl (52.2 g, 165.8 mmol) at 0° C., the mixture wasallowed to stir at r.t. for 5 h. Upon of completion, the mixture waspoured into cold water, extracted with EA, washed brine, dried overanhydrous Na₂SO₄, and concentrated to obtain a residue which waspurified by column chromatography to give 19-2 (67 g, 113.6 mmol, 82.2%yield) as a white solid. ¹H-NMR (400 MHz, DMSO-d₆): δ=11.29 (s, 1H,exchanged with D₂O), 8.21 (d, J=7.2 Hz, 1H), 8.02-8.00 (m, 2H),7.65-7.61 (m, 1H), 7.54-7.50 (m, 2H), 7.38 (d, J 7.2 Hz, 1H), 5.83 (d, J3.6 Hz, 1H, exchanged with D₂O), 5.64 (s, 1H), 4.24 (d, J 13.2 Hz, 1H),4.11 (s, 3H), 3.95 (d, J 12.8 Hz, 1H), 1.09-0.96 (m, 28H). ESI-LCMS: m/z590 [M+H]⁺.

Preparation of Intermediate (19-3):

A solution of 19-2 (54 g, 91.5 mmol) and EtI (285.6 g, 1.8 mol, 146 mL)in toluene (500 mL) was added Ag₂O (63.6 g, 274.6 mmol), the mixture wasstirred at 50° C. for 4 h, filtered and the filtrate was concentrated toobtain a residue which was purified by column chromatography to give19-3 (41.2 g, 66.4 mmol, 72.5% yield) as a whited solid. ¹H-NMR (400MHz, DMSO-d₆): δ=11.34 (s, 1H, exchanged with D₂O), 8.19 (d, J=7.2 Hz,1H), 7.97 (d, J=7.6 Hz, 2H), 7.63 (t, J=7.2 Hz, 1H), 7.51 (t, J=7.6 Hz,2H), 7.38 (d, J 7.2 Hz, 1H), 5.67 (s, 1H), 4.22 (d, J 13.6 Hz, 1H), 4.14(dd, J=4.0, 9.6 Hz, 1H), 4.08 (d, J 9.6 Hz, 1H), 3.95-3.91 (m, 2H), 3.82(dd, J=6.8, 13.6 Hz, 2H), 1.17 (t, J=6.8 Hz, 3H), 1.04-0.94 (m, 28H).ESI-LCMS: m/z 618 [M+H]⁺.

Preparation of Intermediate (19-4):

A solution of 19-3 (41.2 g, 66.4 mmol) in THF (400 mL) was added TBAF (1M in THF) (132.7 mmol, 133 mL), the mixture was stirred at r.t. for 1 h,poured into cold water, extracted with EA, washed brine, dried overanhydrous Na₂SO₄, and concentrated to obtain a solid which was washedwith (PE:EA=1:1) to give 19-4 (19.1 g, 50.6 mmol, 76.3% yield) as awhite solid. ¹H-NMR (400 MHz, DMSO-d₆): δ=11.29 (s, 1H, exchanged withD₂O), 8.57 (d, J=7.6 Hz, 1H), 8.01 (d, J=7.6 Hz, 2H), 7.63 (t, J=7.6 Hz,1H), 7.52 (t, J=8.0 Hz, 2H), 7.36 (d, J 7.6 Hz, 1H), 5.87 (d, J=2.8 Hz,1H), 5.26 (t, J=5.2 Hz, 1H, exchanged with D₂O), 5.05 (d, J=7.2 Hz, 1H,exchanged with D₂O), 4.11-4.06 (m, 1H), 3.94-3.91 (m 1H), 3.86-3.74 (m,3H), 3.71-3.61 (m, 2H), 1.17 (t, J=7.6 Hz, 3H). ESI-LCMS: m/z 376[M+H]⁺.

Preparation of Intermediate (19-5):

A solution of 19-4 (19.1 g, 50.6 mmol) and imidazole (5.2 g, 75.9 mmol)in pyridine (200 mL) was added TBDPSCl (7.1 g, 60.7 mmol), the mixturewas stirred at r.t. for 1 h, poured into cold water, extracted with EA,washed brine, dried over anhydrous Na₂SO₄, and concentrated to obtain asolid which was purified by column chromatography to give 19-5 (20.2 g,32.6 mmol, 64.4% yield) as a white solid. ESI-LCMS: m/z 614 [M+H]⁺.

Preparation of Intermediate (19-6):

A solution of 19-5 (14.0 g, 22.8 mmol) and collidine (6.9 g, 57.0 mmol)in DCM (150 mL) were added DMTrCl (11.6 g, 34.2 mmol) and AgNO₃ (3.9 g,22.8 mmol) under Ar. The mixture was stirred at r.t. for 15 h, filteredand the filtrate was washed with water, dried over anhydrous Na₂SO₄, andconcentrated to give the crude product 19-6 (26.2 g) as a yellow oil.ESI-LCMS: m/z 916 [M+H]⁺.

Preparation of Intermediate (19-7):

A solution of 19-6 (26.2 g (crude)) in THF (200 mL) was added TBAF (1 Min THF) (23 mL), the mixture was stirred at 50° C. for 3 h, poured intocold water, extracted with EA, washed brine, dried over anhydrousNa₂SO₄, and concentrated to obtain a residue which was purified bycolumn chromatography to give 19-7 (11.2 g, 16.5 mmol, 72.37% yield overtwo steps) as a white solid. ¹H-NMR (400 MHz, DMSO-d₆): δ=11.30 (s, 1H,exchanged with D₂O), 8.40 (d, J=7.6 Hz, 1H), 8.02 (d, J=7.6 Hz, 2H),7.65-7.61 (m, 1H), 7.54-7.49 (m, 4H), 7.35-7.30 (m, 6H), 7.26-7.23 (m,1H), 6.89-6.86 (m, 4H), 5.96 (d, J=3.2 Hz, 1H), 5.08 (t, J=4.4 Hz, 1H,exchanged with D₂O), 4.01 (t, J=5.2 Hz, 1H), 3.79-3.78 (m, 1H), 3.73 (d,J=1.2 Hz, 6H), 3.66-3.55 (m 2H), 3.40-3.38 (m, 1H), 3.25-3.17 (m, 1H),2.97-2.95 (m, 1H), 1.17 (t, J=6.8 Hz, 3H). ESI-LCMS: m/z 678 [M+H]⁺.

Preparation of (19-8):

A solution of 19-7 (5.0 g, 7.4 mmol) and DCI (2.2 g, 7.4 mmol) in dryDCM (50 mL) was added CEP[N(iPr)₂]₂ (2.7 g, 8.8 mmol) under Ar. Themixture was stirred at r.t. for 1 h. The reaction was washed with 10%NaHCO₃(aq) and water, dried over Na₂SO₄ and concentrated to obtain thecrude product which was purified Flash-Prep-HPLC. This resulted in togive 19-8 (5.5 g, 6.26 mmol, 84.91% yield) as a white solid. ¹H-NMR (400MHz, DMSO-d₆): δ=11.34 (s, 1H), 8.16 (dd, J=7.6, 39.2 Hz, 1H), 8.03-8.00(m, 2H), 7.65-7.61 (m, 2H), 7.54-7.45 (m, 4H), 7.35-7.24 (m, 8H),6.88-6.82 (m, 4H), 5.89 (dd, J=3.2, 88.8 Hz, 1H), 4.25-3.86 (m, 3H),3.75-3.66 (m, 9H), 3.62-3.38 (m, 4H), 3.26-3.05 (m, 1H), 2.78 (dd,J=5.6, 10.4 Hz, 2H), 1.20-1.11 (m, 9H), 1.04 (dd, J=6.4, 27.6 Hz, 6H).³¹P-NMR (162 MHz, DMSO-d₆): 148.09, 147.84. ESI-LCMS: m/z 878 [M+H]⁺.

Example 16

Preparation of Intermediate (20-2):

To a solution of Thymine (8.15 g, 64.68 mmol, 1.5 eq.) in Acetonitrile(300 mL) with an inert atmosphere of nitrogen, was addedN,O-Bis(trimethylsilyl)acetamide (39 g, 194.2 mmol, 4.5 eq.) at roomtemperature. The resulting solution was stirred for 2 h at 80° C. Thereaction mixture was cooled to 0° C. and 20-1 (30 g, 43.16 mmol, 1.00eq.) was added. Then trimethylsilyl trifluoromethanesulfonate (57.48 g,258.95 mmol, 6.0 eq.) was added dropwise with stirring at 0° C. Theresulting solution was allowed to react, with stirring, for anadditional 1 h at 80° C. The reaction mixture was cooled to 0° C.,quenched by the addition of saturated sodium bicarbonate. The resultingsolution was extracted ethyl acetate and the organic layers combined.The organic layer was washed with water and saturated sodium chloriderespectively. The residue was purified by column chromatography. Thisresulted in 18 g (55%) of 20-2 as a brown solid. MS m/z [M+H]+ (ESI):762.

Preparation of Intermediate (20-3):

To a solution of 20-2 (18 g, 23.9 mmol, 1.00 eq.) in Tetrahydrofuran(180 mL) with an inert atmosphere of nitrogen, was added sodiumhydroxide (47 mL, 2N in water, 4.0 eq.) at 0° C. The resulting solutionwas stirred for 4 h at 0° C. The resulting mixture was extracted withethyl acetate, and the organic layers combined. The organic layer wasdried over anhydrous sodium sulfate and concentrated. The crude productwas purified by Flash-Prep-HPLC. This resulted in 11.4 g (88%) of 20-3as a white solid. MS m/z [M+H]+(ESI): 548.

Preparation of Intermediate (20-4):

To a solution of 20-3 (11.4 g, 20.84 mmol, 1.00 eq.) in Tetrahydrofuran(100 mL) was added 10% Palladium on activated carbon (1.4 g). The flaskwas evacuated and flushed five times with hydrogen. The resultingsolution was stirred for 8 h at room temperature. The solids werefiltered out. The resulting mixture was concentrated under reducedpressure. This resulted in 10 g (92%) of 20-4 as a white solid. MS m/z[M+H]+ (ESI): 522.

Preparation of Intermediate (20-5):

To a solution of 20-4 (10 g, 19.19 mmol, 1.00 eq.) in pyridine (50 mL)with an inert atmosphere of nitrogen, was added1-(chlorodiphenylmethyl)-4-methoxybenzene (8.8 g, 28.79 mmol, 1.5 eq.)at room temperature. The resulting solution was stirred for 12 h at roomtemperature. The reaction was then quenched by the addition of methanol(20 mL). The crude product was purified by Flash-Prep-HPLC. Thefractions (500 mL) was diluted with dichloromethane and dried overanhydrous sodium sulfate. The solid was filtered out. The filtrate wasconcentrated under reduced pressure. This resulted in 12.2 g (80%) of20-5 as a white solid. MS m/z [M−H]− (ESI): 792.

Preparation of Intermediate (20-6):

To a solution of 20-5 (4.2 g, 5.29 mmol, 1.00 eq.) in dichloromethane(40 mL) with an inert atmosphere of nitrogen, was addedTetrabutylammonium fluoride (1M in THF, 13.0 mL, 2.5 eq.) at roomtemperature. The resulting solution was stirred for 10 h at roomtemperature. The crude product was purified by Flash-Prep-HPLC. Thefractions were diluted with dichloromethane and dried over anhydroussodium sulfate. The solid was filtered out. The filtrate wasconcentrated under reduced pressure. This resulted in 2.5 g (85%) of20-6 as a white solid. MS m/z [M−H]− (ESI): 554. 1H NMR: (DMSO-d₆, 300Hz, ppm): δ 11.25 (s, 1H), 7.95 (s, 1H), 7.46 (m, 4H), 7.31-7.11 (m,8H), 6.76 (m, 2H), 5.61 (s, 1H), 5.22 (t, J=3.0 Hz, 1H), 3.95 (m, 1H),3.85-3.61 (m, 6H), 2.85 (m, 1H), 2.45 (d, J=9.0 Hz, 1H), 2.23-2.06 (m,1H), 1.61 (m, 4H), 1.34 (d, J=12.0 Hz, 1H).

Preparation of (20-7):

To a solution of 20-6 (2.5 g, 3.78 mmol, 1.00 eq.) in dichloromethane(20 mL) with an inert atmosphere of nitrogen, was addedBis(diisopropylamino)(2-cyanoethoxy)phosphine (1.7 g, 5.67 mmol, 1.5eq.) at room temperature. To this was added 4,5-Dicyanoimidazole (440mg, 4.15 mmol, 1.10 eq.) at room temperature. The resulting solution wasstirred for 3 h at room temperature and diluted with dichloromethane,washed with water. The crude product was purified by Flash-Prep-HPLC.The fractions were diluted with dichloromethane and dried over anhydroussodium sulfate. The solid was filtered out. The filtrate wasconcentrated under reduced pressure. This resulted in 2.3 g (67%) of20-7 as a white solid. MS m/z [M−H]− (ESI): 754. H-NMR: (DMSO-d₆, 300Hz, ppm): δ 11.31 (s, 1H), 7.76-7.41 (m, 5H), 7.37-7.10 (m, 8H),6.87-6.68 (m, 2H), 5.66 (m, 1H), 4.04 (m, 1H), 3.83 (m, 2H), 3.70 (m,3H), 3.69-3.45 (m, 4H), 3.30-3.00 (m, 1H), 2.88-2.54 (m, 3H), 2.45-2.12(m, 2H), 1.87-1.64 (m, 4H), 1.51 (m, 1H), 1.17 (m, 12H). P NMR (DMSO-d₆,300 Hz, ppm): 152.69, 150.53.

Example 17

Preparation of Intermediate (21-21):

To a solution of(3aR,5S,6S,6aR)-5-(2,2-dimethyl-1,3-dioxolan-4-yl)-2,2-dimethyl-tetrahydro-2H-furo[2,3-d][1,3]dioxol-6-ol(500 g, 1.92 mol, 1.00 eq.) in N,N-dimethylformamide (5000 mL) with aninert atmosphere of nitrogen was added (bromomethyl) benzene (345 g,2.02 mol, 1.05 eq.) at room temperature. Sodium hydride (123 g, 3.08mol, 1.60 eq. 60%) was added for several batches at 0° C. The resultingsolution was stirred for 2 h at 25° C. The reaction was then quenched bythe addition of ammonium chloride (aq). The resulting solution wasextracted with ethyl acetate and the organic layers combined and driedover anhydrous sodium sulfate and concentrated under reduced pressure.This resulted in 670 g (crude) of 21-21 as yellow oil. MS m/z [M+H]+(ESI): 351.

Preparation of Intermediate (21-22):

21-21(500 g, 1.43 mol, 1.00 eq.) was dissolved 80% acetic acid (5000mL). The resulting solution was stirred overnight at room temperature.The resulting mixture was concentrated under reduced pressure. Theresulting solution was diluted with ethyl acetate. The resulting mixturewas washed with sodium bicarbonate (aq). The mixture was dried overanhydrous sodium sulfate and concentrated under reduced pressure. Thisresulted in 440 g (crude) of 21-22 as yellow oil. MS m/z [M+Na]⁺ (ESI):333.

Preparation of Intermediate (21-23):

To a solution of 21-22 (500 g, 1.61 mol, 1.00 eq.) in 1,4-dioxane (5000mL) was added a solution of sodium periodate (345 g, 1.61 mol, 1.00 eq.)in water(5000 mL) dropwise with stirring at 0° C. The resulting solutionwas stirred for 2 h at room temperature. The resulting solution wasdiluted with ethyl acetate. The resulting mixture was washed with water.The mixture was dried over anhydrous sodium sulfate and concentratedunder reduced pressure. This resulted in 400 g (crude) of 21-23 asyellow oil. MS m/z [M+H]+(ESI): 279.

Preparation of Intermediate (21-24):

To a solution of 21-23 (250 g, 898.3 mmol, 1.00 eq.) intetrahydrofuran/water (1250/1250 mL) was added formaldehyde solution(37%, 600 mL) at room temperature. To this was added 2N sodium hydroxide(1500 mL, 3.12 eq.) dropwise with stirring at 0° C. The resultingsolution was stirred overnight at room temperature. The resultingsolution was extracted with ethyl acetate and the organic layerscombined. The resulting mixture was washed with water. The mixture wasdried over anhydrous sodium sulfate and concentrated under reducedpressure. This resulted in 191 g (crude) of 21-24 as yellow oil. MS m/z[M+Na]+ (ESI): 333.

Preparation of Intermediate (21-25):

To a solution of 21-24 (300 g, 966.7 mmol, 1.00 eq.) in pyridine (3000mL) with an inert atmosphere of nitrogen was added benzoyl chloride(409.4 g, 2.91 mol, 3.01 eq.) dropwise with stirring at 0° C. and thenstirred for 2 h at room temperature. The resulting solution was dilutedwith ethyl acetate. The resulting mixture was washed with sodiumbicarbonate (aq). The mixture was dried over anhydrous sodium sulfateand concentrated under reduced pressure. The residue was purified bycolumn chromatography. This resulted in 410 g (82%) of 21-25 as yellowoil.

Preparation of Intermediate (21-26):

To a solution of 21-25 (250 g, 482.11 mmol, 1.00 eq.) in methanol (2500mL) was added 10% Palladium carbon (125 g) and aceticacid (29 g, 482.11mmol 1.00 eq.). Then H₂ (gas) was inserted. The resulting solution wasstirred for 72 h at 50° C. The solids were filtered out. The resultingmixture was concentrated under reduced pressure. The resulting solutionwas diluted with dichloromethane and washed with Sat. sodium bicarbonate(aq). The mixture was dried over anhydrous sodium sulfate andconcentrated under reduced pressure. This resulted in 185 g (90%) of21-26 as light yellow oil.

Preparation of Intermediate (21-27):

To a solution of 21-26 (128 g, 299.06 mmol, 1.00 eq.) in dichloromethane(900 mL) with an inert atmosphere of nitrogen was added pyridine (300mL) at room temperature. To this was added trifluoromethane sulfonicanhydride (109.4 g, 387.9 mmol, 1.30 eq.) dropwise with stirring at −20°C. The resulting solution was stirred for 2 h at −20° C. The resultingsolution was diluted with ethyl acetate. The resulting mixture waswashed with water and brine. The mixture was dried over anhydrous sodiumsulfate and concentrated under reduced pressure. This resulted in 158 g(crude) of 21-27 as yellow oil.

Preparation of Intermediate (21-28):

To a solution of 21-27 (158 g, 281.89 mmol, 1.00 eq.) inN,N-dimethylformamide (1500 mL) with an inert atmosphere of nitrogen wasadded Sodium azide (55 g, 846.02 mmol, 3.00 eq.) and Tetrabutylammoniumiodide (10.4 g, 28.18 mmol, 0.10 eq.) at room temperature. The resultingsolution was stirred overnight at 100° C. The reaction was then quenchedby the addition of water/ice. The resulting solution was extracted withethyl acetate and the organic layers combined. The resulting mixture waswashed with water and brine. The mixture was dried over anhydrous sodiumsulfate and concentrated under reduced pressure. The residue waspurified by column chromatography. This resulted in 58 g (45%) of 21-28as yellow oil.

Preparation of Intermediate (21-29):

To a solution of 21-28 (70 g, 154.4 mmol, 1.00 eq.) in methanol/water(850/150 mL) was added potassiumcarbonate (6.4 g, 46.0 mmol, 0.30 eq.).The resulting solution was stirred for 5 h at 0° C. The pH value of thesolution was adjusted to 7 with 10% hydrochloric acid. The resultingmixture was concentrated under reduced pressure. The resulting solutionwas diluted with ethyl acetate and washed with water. The mixture wasdried over anhydrous sodium sulfate and concentrated under reducedpressure. The residue was purified by column chromatography. Thisresulted in 26.7 g (71%) of 21-29 as a white solid.

Preparation of Intermediate (21-30):

To a solution of 21-29 (12 g, 48.93 mmol, 1.00 eq.) in dichloromethane(360 mL) with an inert atmosphere of nitrogen was followed by theaddition of Triethylamine (14.85 g, 146.75 mmol, 3.00 eq.) at roomtemperature. To this was added tert-butyl (chloro)diphenylsilane (13.5g, 49.12 mmol, 1.00 eq.) dropwise with stirring at 0° C. The resultingsolution was stirred overnight at room temperature. The resultingsolution was diluted with dichloromethane and washed with water. Themixture was dried over anhydrous sodium sulfate and concentrated underreduced pressure. The residue was purified by column chromatography.This resulted in 12.8 g (54%) of 21-30 as a white solid. MS m/z[M+Na]+(ESI): 506.

Preparation of Intermediate (21-31):

To a solution of oxalyl chloride (5.1 mL, 1.50 eq.) in dichloromethane(300 mL) with an inert atmosphere of nitrogen was added dimethylsulphoxide (8.1 mL, 3.00 eq.) dropwise with stirring at −78° C. Theresulting solution was stirred at −78° C. for 30 min. To this was addeda solution of 21-30 (26 g, 43 mmol, 1.00 eq.) in dichloromethane (100mL) dropwise with stirring at −78° C., stirred for an additional 45 minat −78° C. Then triethylamine (23.5 mL, 4.50 eq.) was added, and allowedto react, with stirring, for an additional 1 h at 25° C. The resultingsolution was diluted with dichloromethane, and was washed with hydrogenchloride, saturated sodium bicarbonate, and saturated sodium chloriderespectively. The mixture was dried over anhydrous sodium sulfate,filtered, and concentrated under vacuum. This resulted in 20 g (80%) of21-31 as colorless oil.

Preparation of Intermediate (21-32):

To a solution of 21-31 (20 g, 41.53 mmol, 1.00 eq.) in ether (200 mL)with an inert atmosphere of nitrogen, was added methylmagnesium bromide(3M) (28 mL, 62.30 mmol, 1.50 eq.) dropwise with stirred at −100° C. Theresulting solution was stirred for 2 hours at −100° C. The reaction wasthen quenched by the addition of saturated ammonium chloride (50 mL) anddiluted with ethyl acetate. The resulting mixture was washed with waterand saturated sodium chloride respectively. The organic layer was driedover anhydrous sodium sulfate, filtered, and concentrated under reducedpressure. This resulted in 14.5 g (70%) of 21-32 as colorless oil. MSm/z [M+Na]+ (ESI): 520.

Preparation of Intermediate (21-33):

To a solution of PH21-32 (15 g, 30.14 mmol, 1.00 eq.) in pyridine (150mL) with an inert atmosphere of nitrogen, was added methanesulfonylchloride (4.5 g, 39.28 mmol, 1.30 eq.) dropwise with stirred at 0-5° C.The resulting solution was stirred for 3 hours at 0-5° C. The reactionwas then quenched by the addition of methanol (20 mL) and diluted withethyl acetate. The resulting mixture was washed with water and saturatedsodium chloride. The organic layer was dried over anhydrous sodiumsulfate, filtered, and concentrated under reduced pressure. Thisresulted in 13.8 g (70%) of 21-33 as a brown solid. MS m/z [M+Na]+(ESI): 598.

Preparation of Intermediate (21-34):

To a solution of 21-33 (10 g, 17.37 mmol, 1.00 eq.) in acetic acid (150mL) with an inert atmosphere of nitrogen, was added acetyl acetate (19.5g, 191.01 mmol, 11.00 eq.) and sulfuric acid (170 mg, 1.73 mmol, 0.10eq.). The resulting solution was stirred for 1 hour at room temperature.The resulting solution was diluted with ethyl acetate. The resultingmixture was washed with water and Sat. sodium chloride. The mixture wasdried over anhydrous sodium sulfate and concentrated. The crude productwas purified by Flash-Prep-HPLC. This resulted in 6.5 g (60%) of 21-34as a yellow solid. MS m/z [M+Na]+ (ESI): 642.

Preparation of Intermediate (21-35):

To a solution of 5-methyl-1,2,3,4-tetrahydropyrimidine-2,4-dione (1.5 g,11.90 mmol, 1.50 eq.) in Acetonitrile (125 mL) with an inert atmosphereof nitrogen, was added N, O-Bis(trimethylsilyl)acetamide (5.75 g, 28.19mmol, 3.50 eq.) at room temperature. The resulting solution was stirredfor 2 h at 80° C. To this was added 21-34 (5 g, 8.07 mmol, 1.00 eq.) at0° C., and then trimethylsilyl trifluoromethanesulfonate (2.34 g, 10.53mmol, 1.30 eq.) was added dropwise with stirring at 0° C. The resultingsolution was allowed to react, with stirring, for an additional 6 h at80° C. The reaction mixture was cooled to 0° C., quenched by theaddition of Sat. ammonium chloride. The resulting solution was extractedwith ethyl acetate and the organic layers combined and dried overanhydrous sodium sulfate and concentrated. The residue was purified bycolumn chromatography. This resulted in 4.5 g (81%) of 21-35 as a whitesolid. MS m/z [M+H]+ (ESI): 686.

Preparation of Intermediate (21-36):

To a solution of 21-35 (9 g, 13.12 mmol, 1.00 eq.) in methanol (90 mL)with an inert atmosphere of nitrogen, was added potassium carbonate (5.4g, 38.79 mmol, 3.00 eq.). The resulting solution was stirred for 2 hoursat 65° C. and diluted with ethyl acetate. The resulting mixture waswashed with water and Sat. sodium chloride. The organic layer was driedover anhydrous sodium sulfate and concentrated. The crude product waspurified by Flash-Prep-HPLC. This resulted in 5 g (70%) of 21-36 as awhite solid. MS m/z [M+H]+ (ESI): 548.

Preparation of Intermediate (21-37):

To a solution of 21-36 (6 g, 10.96 mmol, 1.00 eq.) in tetrahydrofuran(60 mL) was added 10% Palladium on activated carbon (2 g). The flask wasevacuated and flushed five times with hydrogen. The resulting solutionwas stirred for 8 hours at room temperature. The solids were filteredout. The resulting mixture was concentrated under reduced pressure. Thisresulted in 5.3 g (93%) of 21-37R and 21-37S as a white solid. Then themixture was separated by Prep-SFC. This resulted in 3.5 g as a whitesolid. MS m/z [M+H]+ (ESI): 522.

Preparation of Intermediate (21-38):

To a solution of 21-37 (3.5 g, 6.71 mmol, 1.00 eq.) in pyridine (35 mL)with an inert atmosphere of nitrogen was added1-(chlorodiphenylmethyl)-4-methoxybenzene (2.7 g, 8.74 mmol, 1.30 eq.)at room temperature. The resulting solution was stirred for 2 hours atroom temperature. The reaction was then quenched by the addition ofmethanol (20 mL). The resulting solution was diluted with ethyl acetate.The resulting mixture was washed with Sat. sodium bicarbonate and brine.The organic layer was dried over anhydrous sodium sulfate andconcentrated. The residue was purified by column chromatography. Thisresulted in 4 g (75%) of 21-38 as a white solid. MS m/z [M−H]⁻ (ESI):792.

Preparation of Intermediate (21-39):

To a solution of 21-38 (4 g, 5.04 mmol, 1.00 eq.) in dichloromethane (40mL) with an inert atmosphere of nitrogen, was added triethylamine (1 g,9.9 mmol, 2.0 eq.) at room temperature. To this was added Triethylaminetrihydrofluoride (0.97 g, 7.56 mmol, 1.50 eq.) at room temperature. Theresulting solution was stirred for 12 hours at room temperature anddiluted with dichloromethane. The resulting mixture was washed withwater. The mixture was dried over anhydrous sodium sulfate andconcentrated. The crude product was purified by Flash-Prep-HPLC. Thisresulted in 2.3 g (80%) of 21-39 as a white solid. 1H NMR (DMSO-d₆, 300Hz, ppm): δ 11.33 (s, 1H), 7.45 (m, 5H), 7.20 (m, 8H), 6.80 (m, 2H),5.35 (t, J=5.1 Hz, 1H), 5.15 (s, 1H), 4.23 (m, 1H), 4.05 (m, 1H), 3.81(m, 1H), 3.73 (s, 3H), 2.92 (d, J=9.7 Hz, 1H), 2.25 (d, J=9.8 Hz, 1H),2.05 (s, 1H), 1.54 (s, 3H), 1.12 (d, J=6.1 Hz, 3H).

Preparation of (21-40):

To a solution of 21-39 in dichloromethane (23 mL) with an inertatmosphere of nitrogen was addedBis(diisopropylamino)(2-cyanoethoxy)phosphine(1.62 g, 5.38 mmol, 1.30eq.) at room temperature. To this was added 4, 5-Dicyanoimidazole (490mg, 4.15 mmol, 1.10 eq.) at room temperature. The resulting solution wasstirred for 1 hour at room temperature and diluted with dichloromethane.Then the mixture was washed with water. The organic layer was dried overanhydrous sodium sulfate and concentrated. The crude product waspurified by Flash-Prep-HPLC. This resulted in 2.3 g (70%) of 21-40 as awhite solid. MS m/z [M−H]− (ESI): 754. 1H NMR (DMSO-d₆, 400 Hz, ppm) δ11.37 (s, 1H), 7.45 (m, 4H), 7.28 (m, 9H), 6.80 (m, 2H), 5.18 (d, J=6.3Hz, 1H), 4.35 (m, 1H), 4.21 (m, 1H), 4.01 (m, 1H), 3.7 (m, 6H), 3.6 (m,1H), 2.90 (m, 3H), 2.33 (m, 1H), 2.13 (m, 1H), 1.59 (d, J=21.6 Hz, 3H),1.20 (m, 15H). P NMR (DMSO-d₆, 400 Hz, ppm): 147.45, 147.08.

Example 18

Preparation of Intermediate (22-2):

To a solution of triphenylphosphane (10.5 g, 40.03 mmol, 2.00 eq.) inToluene (100 mL) with an inert atmosphere of nitrogen, was addeddiisopropyl azodicarboxylate (8.10 g, 40.03 mmol, 2.00 eq.) at roomtemperature. To this was added p-nitrobenzoic acid (6.70 g, 40.03 mmol,2.00 eq.) at 0° C. The resulting solution was stirred for 2 h at roomtemperature. To this was added 22-1 (10 g, 20.09 mmol, 1.00 eq.) at roomtemperature. The resulting solution was allowed to react for anadditional 8 h at 60° C. The reaction mixture was diluted with ethylacetate. The resulting mixture was washed with water and saturatedsodium chloride respectively. The mixture was dried over anhydroussodium sulfate and concentrated. The crude product was purified byFlash-Prep-HPLC. This resulted in 5 g (38%) of 22-2 as a yellow solid.

Preparation of Intermediate (22-3):

To a solution of 22-2 (27.5 g, 42.52 mmol, 1.00 eq.) in methanol (300mL), with an inert atmosphere of nitrogen, was added sodium hydroxide(4.6 g, 85 mmol, 2.0 eq.) at 0° C. The resulting solution was stirredfor 2 hours at 25° C. The pH value of the solution was adjusted to 8with acetic acid. The resulting mixture was concentrated and thendiluted with ethyl acetate. The resulting mixture was washed with waterand saturated sodium chloride. The organic layer was dried overanhydrous sodium sulfate and concentrated. The crude product waspurified by Flash-Prep-HPLC. This resulted in 16.5 g (78%) of 22-3 asyellow oil.

Preparation of Intermediate (22-4):

To a solution of 22-3 (16.5 g, 33.15 mmol, 1.00 eq.) in pyridine (160mL) with an inert atmosphere of nitrogen, was added methanesulfonylchloride (4.95 g, 43.23 mmol, 1.30 eq.) dropwise with stirred at 0-5° C.The resulting solution was stirred for 3 hours at 0-5° C. The reactionwas then quenched by the addition of methanol (30 mL) and diluted withethyl acetate. The resulting mixture was washed with water and Sat.sodium chloride. The mixture was dried over anhydrous calcium chlorideand concentrated under reduced pressure. This resulted in 15.3 g (70%)of 22-4 as a brown solid. MS m/z [M+Na]+ (ESI): 598.

Preparation of Intermediate (22-5):

To a solution of 22-4 (15.3 g, 26.57 mmol, 1.00 eq.) in acetic acid (150mL) with an inert atmosphere of nitrogen, was added acetyl acetate (30g, 292.2 mmol, 11.00 eq.) and sulfuric acid (260 mg, 2.65 mmol, 0.10eq.). The resulting solution was stirred for 1 hour at room temperatureand diluted with ethyl acetate. The resulting mixture was washed withwater and Sat. sodium chloride. The organic layer was dried overanhydrous sodium sulfate and concentrated. The crude product waspurified by Flash-Prep-HPLC. This resulted in 10 g (60%) of 22-5 as ayellow solid. MS m/z [M+Na]+ (ESI): 642.

Preparation of Intermediate (22-6):

To a solution of 5-methyl-1,2,3,4-tetrahydropyrimidine-2,4-dione (3.0 g,23.8 mmol, 1.50 eq.) in Acetonitrile (250 mL) with an inert atmosphereof nitrogen, was added N, O-Bis (trimethylsilyl) acetamide (11.5 g, 56.1mmol, 3.50 eq.) at room temperature. The resulting solution was stirredfor 2 h at 80° C. To this was added 22-5 (10 g, 16.14 mmol, 1.00 eq.) at0° C., and then trimethylsilyl trifluoromethanesulfonate (4.68 g, 21.06mmol, 1.30 eq.) was added dropwise with stirring at 0° C. The resultingsolution was allowed to react, with stirring, for an additional 6 h at80° C. The reaction mixture was cooled to 0° C., quenched by theaddition of Sat. ammonium chloride. The resulting solution was extractedwith ethyl acetate and the organic layers combined and dried overanhydrous sodium sulfate and concentrated. The residue was purified bycolumn chromatography. This resulted in 9 g (81%) of 22-6 as a whitesolid. MS m/z [M+H]+ (ESI): 686.

Preparation of Intermediate (22-7):

To a solution of 22-6 (9 g, 13.12 mmol, 1.00 eq.) in methanol (90 mL)with an inert atmosphere of nitrogen, was added potassium carbonate (5.4g, 38.79 mmol, 3.00 eq.) at room temperature. The resulting solution wasstirred for 2 hours at 65° C. The resulting solution was diluted withethyl acetate. The resulting mixture was washed with water and Sat.sodium chloride. The organic layer was dried over anhydrous sodiumsulfate and concentrated. The crude product was purified byFlash-Prep-HPLC. This resulted in 5 g (70%) of 22-7 as a white solid. MSm/z [M+H]+ (ESI): 548.

Preparation of Intermediate (22-8):

To a solution of 22-7 (5 g, 9.13 mmol, 1.00 eq.) in tetrahydrofuran (50mL) was added 10% Palladium on activated carbon (2 g). The flask wasevacuated and flushed five times with hydrogen. The resulting solutionwas stirred for 8 hours at room temperature. The solids were filteredout. The resulting mixture was concentrated under reduced pressure. Thisresulted in 4.4 g (93%) of 22-8R and 22-8 as a white solid. Then themixture was separated by Prep-SFC. This resulted in 1.9 g 22-8 as awhite solid. MS m/z [M+H]+ (ESI): 522.

Preparation of Intermediate (22-9):

To a solution of 22-8 (1.9 g, 3.64 mmol, 1.00 eq.) in pyridine (20 mL)with an inert atmosphere of nitrogen, was added1-(chlorodiphenylmethyl)-4-methoxybenzene (1.23 g, 4.01 mmol, 1.10 eq.)at room temperature. The resulting solution was stirred for 2 hours atroom temperature. The reaction was then quenched by the addition ofmethanol (10 mL). The resulting solution was diluted with ethyl acetate.The resulting mixture was washed with Sat. sodium bicarbonate and Sat.sodium chloride. The organic layer was dried over anhydrous sodiumsulfate and concentrated. The residue was purified by columnchromatography. This resulted in 2.3 g (80%) of 22-9 as a white solid.MS m/z [M−H]− (ESI): 792.

Preparation of Intermediate (22-10):

To a solution of 22-9 (2.3 g, 2.90 mmol, 1.00 eq.) in dichloromethane(20 mL) with an inert atmosphere of nitrogen, was added triethylamine(723 mg, 7.25 mmol, 2.50 eq.) at room temperature. To this was addedtriethylamine trihydrofluoride (2.8 g, 17.4 mmol, 6.00 eq.) at roomtemperature and the resulting solution was stirred for 2 hours at 25° C.The resulting solution was diluted with dichloromethane, washed withwater and saturated sodium chloride respectively. The organic layer wasdried over anhydrous sodium sulfate, filtered, and concentrated. Thecrude product was purified by Flash-Prep-HPLC. This resulted in 1.7 g(80%) of 22-10 as a white solid. 1H NMR (DMSO-d₆, 400 Hz, ppm) δ11.33(s, 1H), 7.41 (m, 4H), 7.20 (m, 7H), 7.19 (m, 2H), 6.80 (m, 2H), 5.25(t, J=5.2 Hz, 1H), 5.15 (s, 1H), 4.15 (m, 2H), 3.91 (q, J=6.7 Hz, 1H),3.73 (s, 3H), 2.73 (d, J=9.5 Hz, 1H), 2.25 (m, 1H), 2.05 (m, 1H), 1.55(s, 3H), 1.38 (d, J=6.8 Hz, 3H).

Preparation of (22-11):

To a solution of 22-10 (1.77 g, 3.19 mmol, 1.00 eq.) in dichloromethane(20 mL) with an inert atmosphere of nitrogen, was addedBis(diisopropylamino)(2-cyanoethoxy)phosphine(1.35 g, 4.3 mmol, 1.4 eq.)at room temperature. To this was added 4, 5-Dicyanoimidazole (451 mg,3.6 mmol, 1.20 eq.) at room temperature. The resulting solution wasstirred for 2 hours at 25° C. and diluted with dichloromethane. Theresulting mixture was washed with water and saturated sodium chloriderespectively. The organic layer was dried over anhydrous sodium sulfate,filtered, and concentrated. The crude product was purified byFlash-Prep-HPLC. This resulted in 1.82 g (76%) of 22-11 as a whitesolid. MS m/z [M−H]− (ESI): 754. 1H NMR (DMSO-d₆, 400 Hz, ppm) δ 11.4(d, J=9.8 Hz, 1H), 7.40 (m, 4H), 7.28 (m, 8H), 7.07 (m, 1H), 6.79 (m,2H), 5.22 (d, J=4.0 Hz, 1H), 4.38 (m, 1H), 4.27 (m, 1H), 4.01 (q, J=6.7Hz, 1H), 3.7 (m, 7H), 2.82 (m, 1H), 2.70 (m, 1H), 2.13 (m, 1H), 2.32 (d,J=60.2 Hz, 1H), 2.13 (dd, J=28.6 Hz and 6.0 Hz, 1H), 1.60 (d, J=18.0 Hz,3H), 1.45 (t, J=6.9 Hz, 3H), 1.20 (m, 12H). P NMR (DMSO-d₆, 400 Hz,ppm): δ 148.03, 147.53.

Example 19

Preparation of Intermediate (23-2):

To a solution of 23-1 (7.2 g, 9.07 mmol, 1.0 eq.) in Acetonitrile (70mL) with an inert atmosphere of nitrogen, was added Triethylamine (2.75g, 27.24 mmol, 3.0 eq.), 4-(dimethylamino)-pyridin (3.4 g, 27.24 mmol,3.0 eq.) and 2, 4, 6-Triisopropylbenzenesulfonyl chloride (8.226 g,27.24 mmol, 3.0 eq.). The resulting solution was stirred for 12 h atroom temperature. Then ammoniumhydroxide (20 mL) was added and stirredfor 1 hour at room temperature. The resulting solution was extractedwith dichloromethane. The organic layers combined and washed with water,dried over anhydrous sodium sulfate, concentrated. The crude product waspurified by Flash-Prep-HPLC. This resulted in 5.7 g (79%) of 23-2 as ayellow solid MS m/z [M−H]− (ESI): 791.

Preparation of Intermediate (23-3):

To a solution of 23-2 (5.7 g, 7.20 mmol, 1.00 eq.) in Pyridine (70 mL)with an inert atmosphere of nitrogen, was added Benzoyl chloride (1.14g, 1.2 eq.) dropwise at 0° C. The resulting solution was stirred for 12h at room temperature. The reaction was then quenched by the addition ofmethanol. The resulting mixture was concentrated. The residue waspurified by column chromatography. This resulted in 5 g (76%) of 23-3 asa light yellow solid. MS m/z [M−H]− (ESI): 895.

Preparation of Intermediate (23-4):

To a solution of 23-3 (5 g, 5.58 mmol, 1.00 eq.) in Tetrahydrofuran (50mL) was added tetrabutylammonium fluoride (1M in tetrahydrofuran, 11 mL,2.0 eq.). The resulting solution was stirred for 4 h at roomtemperature. The resulting mixture was concentrated. The crude productwas purified by Flash-Prep-HPLC. This resulted in 2.5 g (68%) of 23-4 asa white solid. MS m/z [M−H]− (ESI): 657. 1H NMR (DMSO-d₆, 300 Hz, ppm):δ 13.08 (s, 1H), 8.32-8.18 (m, 2H), 7.70-7.35 (m, 8H), 7.31-7.08 (m,8H), 6.75 (m, 2H), 5.68 (s, 1H), 5.33 (s, 1H), 4.01-3.91 (m, 1H), 3.79(m, 3H), 3.69 (s, 3H), 2.85 (m, 1H), 2.46-2.05 (m, 2H), 1.88 (s, 3H),1.70 (s, 1H), 1.36 (m, 1H).

Preparation of (23-5):

To a solution of 23-4 (2.5 g, 3.80 mmol, 1.00 eq.) in dichloromethane(25 mL) with an inert atmosphere of nitrogen was addedBis(diisopropylamino)(2-cyanoethoxy)phosphine (1.7 g, 5.70 mmol, 1.50eq.) at room temperature. To this was added 4, 5-Dicyanoimidazole (490mg, 4.17 mmol, 1.10 eq.) at room temperature. The resulting solution wasstirred for 3 h at room temperature and diluted with dichloromethane.The resulting mixture was washed with water. The organic layer was driedover anhydrous sodium sulfate and concentrated. The crude product waspurified by Flash-Prep-HPLC. This resulted in 2.9710 g (91%) of 23-5 asa white solid. MS m/z [M+H]+ (ESI): 859. 1H NMR (DMSO-d₆, 300 Hz, ppm):δ 13.07 (s, 1H), 8.15 (m, 2H), 7.70-7.42 (m, 8H), 7.23 (m, 8H), 6.75 (m,2H), 5.81-5.68 (m, 1H), 4.17-3.97 (m, 1H), 3.96-3.55 (m, 9H), 3.10-2.55(m, 4H), 2.38-2.19 (m, 1H), 1.98 (m, 4H), 1.81-1.38 (m, 2H), 1.20 (m,12H). P NMR (DMSO-d₆, 300 Hz, ppm): 148.11, 145.59.

Example 20

Preparation of Intermediate (24-2):

To a solution of(3aR,5S,6S,6aR)-5-(2,2-dimethyl-1,3-dioxolan-4-yl)-2,2-dimethyl-tetrahydro-2H-furo[2,3-d][1,3]dioxol-6-ol(500 g, 1.92 mol, 1.00 eq.) in N,N-dimethylformamide (5000 mL) with aninert atmosphere of nitrogen, was added (bromomethyl) benzene (345 g,2.02 mol, 1.05 eq.) at room temperature. Sodium hydride (123 g, 3.0 mol,1.60 eq.) was added for several batches at 0° C. The resulting solutionwas stirred for 2 h at 25° C. The reaction was then quenched by theaddition of Saturated ammoniumchloride (2000 mL). The resulting solutionwas extracted with ethyl acetate and the organic layers combined, driedover anhydrous sodium sulfate, filtered and concentrated. This resultedin 670 g (crude) of 24-2 as yellow oil. This crude product was used inthe next step without further purification. MS m/z [M+H]+ (ESI): 351.

Preparation of Intermediate (24-3):

24-2 (300 g, 856.14 mmol, 1.00 eq.) was dissolved acetic acid (80% inwater, 3000 mL). The resulting solution was stirred overnight at roomtemperature. The resulting mixture was concentrated and diluted withethyl acetate. The resulting mixture was washed with Saturated sodiumbicarbonate respectively. The organic phase was dried over anhydroussodium sulfate, filtered and concentrated. This resulted in 237 g(crude) of 24-3 as yellow oil. This crude product was used in the nextstep without further purification. MS m/z [M+Na]+ (ESI): 333.

Preparation of Intermediate (24-4):

To a solution of 24-3 (200 g, 644.44 mmol, 1.00 eq.) in 1,4-dioxane(2000 mL) was added a solution of sodium periodate (138 g, 644.44 mmol,1.00 eq.) in water(2000 mL) dropwise with stirring at 0° C. Theresulting solution was stirred for 2 h at room temperature. Theresulting solution was diluted with ethyl acetate and washed with water.The organic phase was dried over anhydrous sodium sulfate, filtered andconcentrated. This resulted in 160 g (crude) of 24-4 as yellow oil. Thiscrude product was used in the next step without further purification. MSm/z [M+H]+ (ESI): 279.

Preparation of Intermediate (24-5):

To a solution of 24-4 (200 g, 718.64 mmol, 1.00 eq.) intetrahydrofuran/water (1000 mL/1000 mL) was added Formaldehyde Solution(37%, 480 mL) at room temperature. To this was added 2N sodium hydroxide(1200 mL, 3.12 eq.) dropwise with stirring at 0° C. The resultingsolution was stirred overnight at room temperature. The reaction mixturewas extracted with ethyl acetate and the organic layers combined. Theorganic layer was washed with water. The organic phase was dried overanhydrous sodium sulfate, filtered and concentrated. This resulted in152.8 g (crude) of 24-5 as yellow oil. This crude product was used inthe next step without further purification. MS m/z [M+Na]+ (ESI): 333.

Preparation of Intermediate (24-6):

To a solution of 24-5 (500 g, 1.61 mmol, 1.00 eq.) in methanol/Aceticacid (5000/500 mL) in a 20-L pressure tank reactor with an inertatmosphere of nitrogen, was added 10% Palladium on activated carbon (250g). The pressure tank was evacuated and flushed five times withhydrogen. The resulting solution was stirred for 4 days at 50° C. (8atm). The solids were filtered out. The resulting mixture wasconcentrated. The residue was purified by column chromatography. Thisresulted in 280 g (79%) of 24-6 as a white solid.

Preparation of Intermediate (24-7):

To a solution of 24-6 (200 g, 908.2 mmol, 1.00 eq.) in dichloromethane(6000 mL) with an inert atmosphere of nitrogen, was added benzaldehyde(436.0 g, 2722.9 mmol, 3.00 eq.) dropwise with stirring at 0° C. Thencamphorsulfonic acid (2 g) was added at 0° C. The resulting solution wasstirred overnight at room temperature. The reaction was then quenched bythe addition of 2000 mL Saturated sodium bicarbonate. The resultingsolution was extracted with dichloromethane and the organic layerscombined. The organic phase was dried over anhydrous sodium sulfate,filtered and concentrated. The residue was purified by columnchromatography. This resulted in 13.5 g (48%) of 24-7 as a white solid.MS m/z [M+Na]+ (ESI): 331. 1H NMR (DMSO-d₆, 400 Hz, ppm): δ 7.44-7.30(m, 6H), 5.97 (d, J=4.1 Hz, 1H), 5.46 (s, 1H), 5.06 (t, J=5.4 Hz, 1H),4.64-4.52 (m, 2H), 4.14 (d, J=13.0 Hz, 1H), 4.08-3.93 (m, 1H), 3.74-3.65(m, 1H), 3.38 (m, 1H), 1.46 (s, 3H), 1.23 (s, 3H).

Preparation of Intermediate (24-8):

To a solution of oxalyl chloride (30.9 g, 243.31 mmol, 1.50 eq.) indichloromethane (1000 mL) with an inert atmosphere of nitrogen, wasadded of dimethyl sulfoxide (37.9 g, 485.09 mmol, 3.00 eq.) dropwisewith stirring at −78° C. The resulting solution was stirred for 30 minat −78° C. To this was added a solution of 24-7 (50 g, 162.17 mmol, 1.00eq.) in dichloromethane (200 mL) dropwise with stirring at −78° C. Theresulting solution was allowed to react, with stirring, for anadditional 1.5 h at −78° C. Then was added triethylamine (73.9 g, 730.31mmol, 4.50 eq.) dropwise with stirring at −78° C. The resulting solutionwas allowed to react, with stirring, for an additional 2 h at −78° C.The resulting solution was diluted with dichloromethane and washed with5% hydrochloric acid, Saturated sodiumbicarbonate, and Saturated sodiumchloride. The organic phase was dried over anhydrous sodium sulfate,filtered and concentrated. This resulted in 49 g (crude) of 24-8 as awhite solid.

This crude product was used in the next step without furtherpurification. MS m/z [M+H]+(ESI): 307.

Preparation of Intermediate (24-9):

To a solution of methyltriphenylphosphonium bromide (139.8 g, 2.00 eq.)in tetrahydrofuran (600 mL) with an inert atmosphere of nitrogen, wasadded Potassium tert-butoxide (43.8 g, 390.33 mmol, 2.00 eq.). Theresulting solution was stirred for 30 min at room temperature. To this asolution of 24-8 (60 g, 195.87 mmol, 1.00 eq.) in tetrahydrofuran (150mL) was added dropwise with stirring at 0° C. The resulting solution wasallowed to react, with stirring, for an additional 2 h at roomtemperature. The reaction was then quenched by the addition of SaturatedAmmonium chloride (400 mL). The resulting solution was extracted withethyl acetate and the organic layers combined. The organic layer waswashed with water, and Saturated sodium chloride. The organic phase wasdried over anhydrous sodium sulfate, filtered and concentrated. Theresidue was purified by column chromatography. This resulted in 38.7 g(65%) of 24-9 as a white solid. MS m/z [M+H]+ (ESI): 305.

Preparation of Intermediate (24-10):

To a solution of 24-9 (60 g, 197.16 mmol, 1.00 eq.) in tetrahydrofuran(600 mL) with an inert atmosphere of nitrogen, was added9-Borabicyclo[3.3.1]nonane (0.5M in tetrahydrofuran, 789 mL, 2.00 eq.).The resulting solution was stirred overnight at 35° C. Then sodiumhydroxide (2N in water, 591 mL, 6.00 eq.) was added at room temperature.To this was added hydrogen peroxide (30%, 120 mL, 6.00 eq.) dropwisewith stirring at room temperature. The resulting solution was allowed toreact. The resulting solution was extracted with dichloromethane and theorganic layers combined. The organic phase was dried over anhydroussodium sulfate, filtered and concentrated. The residue was purified bycolumn chromatography. This resulted in 43.5 g (68%) of 24-10 as a whitesolid. MS m/z [M+Na]+ (ESI): 345.

Preparation of Intermediate (24-11):

To a solution of 24-10 (100 g, 310.2 mmol, 1.00 eq.) in pyridine (1000mL) with an inert atmosphere of nitrogen, was added benzoyl chloride(130 g, 924.8 mmol, 3.00 eq.) dropwise with stirring at 0° C. Theresulting solution was stirred for 3 h at room temperature. The reactionwas then quenched by the addition of methanol (200 mL). The resultingmixture was concentrated. The solid was diluted with ethyl acetate. Theresulting mixture was washed with water and Saturated sodium chloride.The organic phase was dried over anhydrous sodium sulfate, filtered andconcentrated. The residue was purified by column chromatography. Thisresulted in 110 g (83%) of 24-11 as a white solid. MS m/z [M+Na]+(ESI):449.

Preparation of Intermediate (24-12):

To a solution of 24-11 (200 g, 469 mmol, 1.00 eq.) inmethanol/tetrahydrofuran (500/100 mL) was added 10% Palladium onactivated carbon (100 g). The flask was evacuated and flushed five timeswith hydrogen. The resulting solution was stirred for 48 h at 40° C. Thesolids were filtered out. The resulting mixture was concentrated. Theresidue was purified by column chromatography. This resulted in 35 g(88%) of 24-12 as colorless oil. MS m/z [M+Na]+ (ESI): 361.

Preparation of Intermediate (24-13):

To a solution of 24-12 (50 g, 147.77 mmol, 1.00 eq.) and imidazole (19.5g, 286.43 mmol, 2.00 eq.) in N,N-dimethylformamide (500 mL) with aninert atmosphere of nitrogen, was added of tert-Butyldiphenylsilane(44.5 g, 1.10 eq.) dropwise with stirring at 0° C. The resultingsolution was stirred for 2 h at room temperature. The reaction was thenquenched by the addition of methanol (100 mL). The resulting mixture wasconcentrated. The residue was dissolved in dichloromethane. Theresulting mixture was washed with water and Saturated sodium chloride.The organic phase was dried over anhydrous sodium sulfate, filtered andconcentrated. The residue was purified by column chromatography. Thisresulted in 60 g (70%) of 24-13 as a solid. MS m/z [M+Na]+ (ESI): 599.1H NMR (DMSO-d₆, 300 Hz, ppm): 7.90-7.80 (m, 2H), 7.72-7.54 (m, 5H),7.49-7.30 (m, 8H), 5.85 (d, J=4.4 Hz, 1H), 5.59 (d, J=4.9 Hz, 1H), 4.54(m, 1H), 4.37 (t, J=7.0 Hz, 2H), 4.09 (m, 1H), 3.29 (s, 2H), 2.22 (m,2H), 1.42 (s, 3H), 1.21 (s, 3H), 0.96 (s, 9H.

Preparation of Intermediate (24-14):

To a solution of 24-13 (64 g, 110.97 mmol, 1.00 eq.) indichloromethane/pyridine(448/134 mL) with an inert atmosphere ofnitrogen, was added Trifluoromethanesulfonic anhydride (46.9 g, 166.23mmol, 1.50 eq.) dropwise with stirring at 0° C. The resulting solutionwas stirred for 2 h at 0° C. The resulting solution was diluted withdichloromethane. The resulting mixture was washed with water andSaturated sodium chloride. The organic phase was dried over anhydroussodium sulfate, filtered and concentrated. This resulted in 78 g (crude)of 24-14 as a solid. This crude product was used in the next stepwithout further purification. MS m/z [M+Na]+ (ESI): 731.

Preparation of Intermediate (24-15):

To a solution of 24-14 (78 g, 110.04 mmol, 1.00 eq.) andTetrabutylammonium iodide (4 g, 10.83 mmol, 0.10 eq.) inN,N-dimethylformamide (800 mL) with an inert atmosphere of nitrogen, wasadded Sodium azide (21.5 g, 330.72 mmol, 3.00 eq.). The resultingsolution was stirred overnight at 100° C. The reaction was then quenchedby the addition of ice water (200 mL). The resulting solution wasdiluted with ethyl acetate. The resulting mixture was washed with waterand Saturated sodium chloride. The organic phase was dried overanhydrous sodium sulfate, filtered and concentrated. The residue waspurified by column chromatography. This resulted in 45 g (68%) of 24-15as brown oil. MS m/z [M+Na]+(ESI): 624.

Preparation of Intermediate (24-16):

To a solution of 24-15 (91 g, 151.22 mmol, 1.00 eq.) in methanol(900 mL)with an inert atmosphere of nitrogen, was added sodium methoxide (30% inmethanol, 50 mL, 2.00 eq.). The resulting solution was stirred for 2 hat room temperature. The resulting solution was extracted with ethylacetate and the organic layers combined. The organic layer was washedwith water and Saturated sodium chloride. The organic phase was driedover anhydrous sodium sulfate, filtered and concentrated. The residuewas purified by column chromatography. This resulted in 55 g (73%) of24-16 as a white solid. MS m/z [M+Na]+(ESI): 520.

Preparation of Intermediate (24-17):

To a solution of 24-16 (56 g, 112.53 mmol, 1.00 eq.) and triethylamine(67.2 g, 664.10 mmol, 6.00 eq.) in dichloromethane(825 mL) with an inertatmosphere of nitrogen, was added 4-dimethylaminopyridine (1 g, 8.19mmol, 0.31 eq.) and 4-toluene sulfonyl chloride (31.5 g, 165.22 mmol,1.50 eq.) in order at room temperature. The resulting solution wasstirred for 2 h at room temperature. The resulting solution was dilutedwith dichloromethane. The resulting mixture was washed with water andSaturated sodium chloride. The organic phase was dried over anhydroussodium sulfate, filtered and concentrated. The residue was purified bycolumn chromatography. This resulted in 55 g (75%) of 24-17 as colorlessoil. MS m/z [M+Na]+ (ESI): 674.

Preparation of Intermediate (24-18):

To a solution of 24-17 (100 g, 153.4 mmol, 1.00 eq.) in acetic acid(1000mL) with an inert atmosphere of nitrogen, were added acetyl acetate(17.2 g, 168.48 mmol, 11.00 eq.) and sulfuric acid (300 mg, 3.08 mmol,0.20 eq.). The resulting solution was stirred for 2 h at roomtemperature. The reaction was then quenched by the addition of ice water(600 mL). The resulting solution was extracted with ethyl acetate. Theresulting mixture was washed water and Saturated sodium chloride. Theorganic phase was dried over anhydrous sodium sulfate, filtered andconcentrated. The residue was purified by column chromatography. Thisresulted in 52 g (49%) of 24-18 as a brown solid. MS m/z [M+Na]+ (ESI):718.

Preparation of Intermediate (24-19):

To a solution of 6-N-benzoyladenine(6.7 g, 1.50 eq.) in acetonitrile(100 mL) with an inert atmosphere of nitrogen, was addedN,O-Bis(trimethylsilyl)acetamide (18.9 g, 92.91 mmol, 5.00 eq.). Theresulting solution was stirred for 1 h at 85° C. This was followed bythe addition of 24-18 (13 g, 18.68 mmol, 1.00 eq.) in acetonitrile (100mL) at 0° C. To this trifluoromethanesulfonic acid trimethylsilyl ester(20.7 g, 92.91 mmol, 5.00 eq.) was added dropwise a at 0° C. Theresulting solution was allowed to react, with stirring, for anadditional 4 h at 85° C. The reaction was then quenched by the additionof Saturated sodium bicarbonate(100 mL). The resulting solution wasextracted with ethyl acetate and the organic layers combined. Theorganic layer was washed with water and Saturated sodium chloride. Theorganic phase was dried over anhydrous sodium sulfate, filtered andconcentrated. The crude product was purified by Flash-Prep-HPLC. Thisresulted in 8 g (49%) of 24-19 as a white solid. MS m/z [M+H]+ (ESI):875.

Preparation of Intermediate (24-20):

To a solution of 24-19 (7.4 g, 8.46 mmol, 1.00 eq.) intetrahydrofuran(55 mL) with an inert atmosphere of nitrogen, was added asolution of lithium hydroxide (1.01 g, 42.17 mmol, 5.00 eq.) in water(25 mL) at 0° C. The resulting solution was stirred for 4 h at roomtemperature. The resulting solution was diluted with ethyl acetate. Theresulting mixture was washed with water and Saturated sodium chloride.The organic phase was dried over anhydrous sodium sulfate, filtered andconcentrated. The residue was purified by column chromatography. Thisresulted in 4.3 g (77%) of 24-20 as a white solid. MS m/z [M+H]+(ESI):661.

Preparation of Intermediate (24-21):

To a solution of 24-20 (4 g, 6.05 mmol, 1.00 eq.) in tetrahydrofuran (40mL), was added 10% Palladium on activated carbon (1.2 g). The flask wasevacuated and flushed five times with hydrogen. The resulting solutionwas stirred for 3 h at 25° C. The solids were filtered out. Theresulting mixture was concentrated. This resulted in 3.8 g (99%) of24-21 as a white solid. MS m/z [M+H]+ (ESI): 635.

Preparation of Intermediate (24-22):

To a solution of 24-21 (3.8 g, 5.99 mmol, 1.00 eq.) in pyridine(38 mL)with an inert atmosphere of nitrogen, was added 4-Methoxytriphenylmethylchloride (2.0 g, 6.59 mmol, 1.10 eq.) at 0° C. The resulting solutionwas stirred for 3 h at 25° C. The reaction was then quenched by theaddition of methanol (5 mL). The resulting mixture was concentrated. Theresidue was dissolved in dichloromethane. The resulting mixture waswashed with water and Saturated sodium chloride. The organic phase wasdried over anhydrous sodium sulfate, filtered and concentrated. Thecrude product was purified by Flash-Prep-HPLC. This resulted in 2.7 g(50%) of 24-22 as a white solid. MS m/z [M+H]+ (ESI): 908.

Preparation of Intermediate (24-23):

To a solution of 24-22(2.7 g, 2.98 mmol, 1.00 eq.) in dichloromethane(27 mL) with an inert atmosphere of nitrogen, was addedtriethylamine(750 mg, 7.41 mmol, 2.50 eq.) and triethylaminetrihydrofluoride (2.87 g, 17.80 mmol, 6.00 eq.) in order. The resultingsolution was stirred for 2 h at 25° C. The resulting solution wasdiluted with dichloromethane. The resulting mixture was washed withwater and Saturated sodium chloride. The organic phase was dried overanhydrous sodium sulfate, filtered and concentrated. The crude productwas purified by Flash-Prep-HPLC. This resulted in 1.8 g (90%) of 24-23as a white solid. MS m/z [M+H]+ (ESI): 669. 1H NMR (DMSO-d₆, 300 Hz,ppm): δ 11.18 (s, 1H), 8.63 (d, J=13.7 Hz, 2H), 8.13-8.03 (m, 2H),7.71-7.49 (m, 3H), 7.42-7.29 (m, 4H), 7.23-6.92 (m, 8H), 6.62-6.51 (m,2H), 6.25 (s, 1H), 5.27 (t, J=4.3 Hz, 1H), 4.04 (m, J=12.0 Hz, 4.6 Hz,1H), 3.88 (t, J=5.5 Hz, 3H), 3.57 (s, 3H), 3.04 (d, J=10.6 Hz, 1H), 2.41(d, J=10.8 Hz, 1H), 2.20 (m, J=12.2 Hz, 7.7 Hz, 1H), 1.57 (d, J=2.5 Hz,1H), 1.44 (d, J=11.7 Hz, 1H).

Preparation of (24-24):

To a solution of 24-23 (1.8 g, 2.69 mmol, 1.00 eq.) in dichloromethane(20 mL) with an inert atmosphere of nitrogen, was addedBis(diisopropylamino)(2-cyanoethoxy)phosphine (1.26 g, 1.40 eq.) andadded 4, 5-Dicyanoimidazole (422 mg, 1.20 eq.) in order at 0° C. Theresulting solution was stirred for 1 h at 25° C. The resulting solutionwas diluted with dichloromethane. The resulting mixture was washed withSaturated sodium bicarbonate and Saturated sodium chloride. The organicphase was dried over anhydrous sodium sulfate, filtered andconcentrated. The crude product was purified by Flash-Prep-HPLC. Thisresulted in 2.1 g (77%) of 24-24 as a white solid. MS m/z [M+H]+ (ESI):869. 1H NMR (DMSO-d₆, 300 Hz, ppm): δ 11.20 (s, 1H), 8.62 (d, J=17.2 Hz,1H), 8.40 (m, 1H), 8.13-8.03 (m, 2H), 7.65 (m, 1H), 7.56 (m, 2H),7.47-7.33 (m, 4H), 7.21 (t, J=8.3 Hz, 2H), 7.15-6.92 (m, 6H), 6.59 (d,J=8.5 Hz, 2H), 6.31 (d, J=3.6 Hz, 1H), 4.22-4.02 (m, 1H), 4.00-3.37 (m,9H), 3.04 (d, J=10.6 Hz, 1H), 2.92-2.67 (m, 2H), 2.55 (d, J=11.3 Hz,1H), 2.31 (m, 1H), 1.73-1.47 (m, 2H), 1.27-0.99 (m, 13H), 0.93-0.76 (m,1H). P-NMR (DMSO-d₆, 300 Hz, ppm): δ148.21, 147.04.

Example 21

Preparation of Intermediate (25-2):

To a solution of N-2-acetylguanine (12.4 g, 56.36 mmol, 3.00 eq.) in 1,2-dichloroethane (200 mL) with an inert atmosphere of nitrogen, wasadded N, O-Bis (trimethylsilyl) acetamide (19 g, 93.6 mmol, 5.00 eq.) atroom temperature. The resulting solution was stirred for 3 h at 85° C.To this was added 25-1 (13 g, 18.68 mmol, 1.00 eq.) at 0° C., and thentrimethylsilyl trifluoromethanesulfonate (20.8 g, 93.58 mmol, 5.00 eq.)was added dropwise with stirring at 0° C. The resulting solution wasallowed to react, with stirring, for an additional 2 h at 80° C. Thereaction mixture was cooled to 0° C., quenched by the addition ofsaturated ammonium chloride (80 mL). The resulting solution wasextracted with ethyl acetate and the organic layers combined and driedover anhydrous sodium sulfate, filtered, and concentrated. The residuewas purified by column chromatography. This resulted in 5.6 g (35%) of25-2 as a white solid. MS m/z [M+H]+ (ESI): 857.

Preparation of Intermediate (25-3):

To a solution of 25-2 (5.6 g, 6.53 mmol, 1.00 eq.) in tetrahydrofuran(56 mL) with an inert atmosphere of nitrogen, was added 1M sodiumhydroxide (56 mL) at 0° C. The resulting solution was stirred for 1 h at0° C. The pH value of the solution was adjusted to 7 with acetic acid.The resulting solution was diluted with ethyl acetate. The resultingmixture was washed with water and saturated sodium chloriderespectively. The organic layer was dried over anhydrous sodium sulfate,filtered, and concentrated. The crude product was purified byFlash-Prep-HPLC. This resulted in 2.9 g (69%) of 25-3 as a white solid.

Preparation of Intermediate (25-4):

To a solution of 25-3 (2.9 g, 4.51 mmol, 1.00 eq.) in tetrahydrofuran(30 mL) was added 10% Palladium on activated carbon (1.2 g). The flaskwas evacuated and flushed five times with hydrogen. The resultingsolution was stirred for 5 hours at room temperature. The solids werefiltered out. The resulting mixture was concentrated under reducedpressure. This resulted in 2.5 g (90%) of 25-4 as a white solid.

Preparation of Intermediate (25-5):

To a solution of 25-4 (2.5 g, 4.05 mmol, 1.00 eq.) in pyridine (25 mL)with an inert atmosphere of nitrogen, was added1-(chlorodiphenylmethyl)-4-methoxybenzene (1.9 g, 6.15 mmol, 1.50 eq.)and 4-dimethylaminopyridine (150 mg, 1.23 mmol, 0.30 eq.) in order atroom temperature. The resulting solution was stirred for 2 h at roomtemperature and then quenched by the addition of methanol (10 mL). Theresulting solution was diluted with ethyl acetate. The resulting mixturewas washed with water and saturated sodium chloride respectively. Theorganic layer was dried over anhydrous sodium sulfate, filtered, andconcentrated. The crude product was purified by Flash-Prep-HPLC. Thisresulted in 2.7 g (75%) of 25-5 as a white solid.

Preparation of Intermediate (25-6):

To a solution of 25-5 (2.7 g, 3.04 mmol, 1.00 eq.) in dichloromethane(30 mL) with an inert atmosphere of nitrogen was added triethylamine (1g, 9.88 mmol, 3.00 eq.) at room temperature. To this was addedtriethylamine trihydrofluoride (1.5 g, 9.32 mmol, 3.0 eq.) at roomtemperature. The resulting solution was stirred for 2 hours at 25° C.and diluted with dichloromethane. The resulting mixture was washed withwater and saturated sodium chloride respectively. The organic layer wasdried over anhydrous sodium sulfate, filtered, and concentrated. Thecrude product was purified by Flash-Prep-HPLC. This resulted in 1.7 g(86%) of 25-6 as a white solid. MS m/z [M+H]+ (ESI): 651. 1H NMR(DMSO-d₆, 400 Hz, ppm) δ 12.16 (s, 1H), 11.77 (s, 1H), 8.15 (s, 1H),7.42 (m, 4H), 7.15 (m, 8H), 6.56 (d, J=8.4 Hz, 2H), 5.97 (s, 1H), 5.22(t, J=4.3 Hz, 1H), 4.01 (m, 1H), 3.85 (m, 2H), 3.73 (m, 1H), 3.60 (s,3H), 3.01 (m, 1H), 2.82 (m, 1H), 2.39 (d, J=10.9 Hz, 1H), 2.17 (m, 1H),1.58 (d, J=2.6 Hz, 1H), 1.41 (m, 1H), 1.18 (d, J=6.8 Hz, 3H), 1.12 (d,J=6.8 Hz, 3H.

Preparation of (25-7):

To a solution of 25-6 (1.7 g, 2.61 mmol, 1.00 eq.) in dichloromethane(17 mL) with an inert atmosphere of nitrogen was added Bis(diisopropylamino) (2-cyanoethoxy) phosphine (950 mg, 3.16 mmol, 1.30eq.) at room temperature. To this was added 4, 5-Dicyanoimidazole (340mg, 2.91 mmol, 1.10 eq.) at room temperature. The resulting solution wasstirred for 2 hours at 25° C. and diluted with dichloromethane. Theresulting mixture was washed with water and saturated sodium chloriderespectively. The organic layer was dried over anhydrous sodium sulfate,filtered, and concentrated. The crude product was purified byFlash-Prep-HPLC. This resulted in 1.8 g (81%) of 25-7 as a white solid.MS m/z [M+H]+ (ESI): 851. H NMR (DMSO-d₆, 400 Hz, ppm) δ 12.18 (s, 1H),11.80 (s, 1H), 7.98 (d, J=64.4 Hz, 1H), 7.47 (m, 4H), 7.15 (m, 8H), 6.56(m, 2H), 6.01 (d, J=10.0 Hz, 1H), 3.95 (m, 3H), 3.77 (m, 2H), 3.66 (m,1H), 3.59 (d, J=9.8 Hz, 3H), 3.38 (m, 2H), 2.91 (m, 4H), 2.30 (m, 1H),1.55 (m, 2H), 1.15 (m, 14H), 1.04 (d, J=6.7 Hz, 4H). P NMR (DMSO-d₆, 400Hz, ppm): 148.62, 147.09.

Example 22

The synthesis of the 2′-O-vinyl adenosine analog 26-8 was achieved asshown in scheme 26. Intermediate 6-2 was treated with NH₃/MeOH to affordcompound 26-1. To avoid side reactions of the vinyl moiety with theazide, the C3′-azide was reduced with a reducing agent such as H₂, Pd/Cto afford intermediate 26-2 whose free 3′-NH₂ was protected with MMTrprotecting group to form 26-3. After deprotection of the N6 position ofthe base to give 26-4, alkylation was carried out with allyl bromide toobtain 26-5. This approach used reduction of the C3′-azide prior toalkylation to avoid side reactions of the vinyl moiety with the azide.Reprotection of N6 position with benzyl chloride afforded compound 26-6that was 5′-deprotected with Py/NaOH/MeOH/H₂O to give 26-7. Standardphosphitylation conditions were used to achieve target compound 26-8.

Preparation of Intermediate 26-1:

To a solution of 6-2 (32.6 g, 58.6 mmol) in methanol (500 mL) was addedNH₃.H₂O (50 mL) at 0° C. The reaction mixture was stirred at 0° C. for 2h and concentrated, the residue was dissolved in EA, washed with brine,dried over anhydrous Na₂SO₄, the solvent was removed and the residue waspurified on silica gel to give 26-1 (28.5 g, 55.4 mmol, 94.62% yield) asa yellow solid. ESI-LCMS: m/z 515 [M+H]⁺.

Preparation of Intermediate 26-2:

To a solution of 26-1 (28.5 g, 55.4 mmol) in THF (400.00 mL) was addedPd/C (3.0 g), the mixture was stirred at r.t. for 6 h under H₂. Themixture was filtered and the filtrate was concentrated to afford 26-2(21.5 g, 44.1 mmol, 79.60% yield) as a gray solid. ESI-LCMS: m/z 489[M+H]⁺.

Preparation of Intermediate 26-3:

MMTrCl (20.4 g, 66.1 mmol) was added to a solution of 26-2 (21.5 g, 44.1mmol) in pyridine (300 mL). The mixture was stirred at room temperaturefor 1 h. The reaction was quenched with MeOH and concentrated toobtained a residue which was purified on silica gel to give 26-3 (22.0g, 28.9 mmol, 65.53% yield) as a white solid. ESI-LCMS: m/z 761 [M+H]⁺.

Preparation of Intermediate 26-4:

To a solution of 26-3 (22.0 g, 28.9 mmol) in a mixture of pyridine (200mL) and AcOH (50 mL), was added hydrazine hydrate (80%) (173.5 mmol, 10mL), and the reaction was stirred at room temperature for 15 h. Thereaction was poured into ice water, extracted with EA, washed with brineand dried over anhydrous Na₂SO₄, concentrated to obtain the crudeproduct which was purified on silica gel to give 26-4 (18.0 g, 27.4mmol, 94.79% yield) as a white solid. ESI-LCMS: m/z 657 [M+H]+.

Preparation of Intermediate 26-5:

Ag₂O (9.5 g, 41.1 mmol, 1.33 mL) and NaI (8.2 g, 54.8 mmol) were addedto a solution of 26-4 (18 g, 27.4 mmol) and vinyl bromide (6.6 g, 54.8mmol) in dry DMF (250 mL) were added the mixture was stirred at roomtemperature for 1.5 h. The reaction was poured into ice water, extractedwith EA, washed with brine and dried over anhydrous Na₂SO₄, concentratedto obtain the crude product which was purified by recrystallization(PE:EA=1:1) to give 26-5 (12.1 g, 17.3 mmol, 63.36% yield) as a whitesolid. ¹H-NMR (400 MHz, DMSO-d₆): δ=7.99 (d, J 2.0 Hz, 1H), 7.53-7.48(m, 6H), 7.40-7.37 (m, 2H), 7.28 (br s, 2H, exchanged with D₂O),7.21-7.14 (m, 6H), 7.09-7.04 (m, 2H), 6.68 (d, J 9.2 Hz, 2H), 5.93 (s,1H), 5.87-5.77 (m, 1H), 5.22-5.16 (m, 1H), 5.10-5.07 (m, 1H), 4.81-4.78(m, 1H), 4.68 (dd, J 3.2, 12.4 Hz, 1H), 4.05-3.99 (m, 1H), 3.94-3.89 (m,1H), 3.53 (s, 3H), 3.22-3.18 (m, 1H), 2.92 (d, J 10.8 Hz, 1H, exchangedwith D₂O), 2.49 (d, J 4.8 Hz, 1H), 2.37 (s, 3H). ESI-LCMS: m/z 697[M+H]⁺.

Preparation of Intermediate 26-6:

BzCl (4.8 g, 34.4 mmol) was added dropwise to a solution of 26-5 (12 g,17.2 mmol) in pyridine (120 mL) at 0° C. After the mixture was stirredfor 1 h at r.t., 300 mL H₂O and 500 mL EA were added to separate thesolution, the aqueous phase was extracted by EA. The combined organiclayers were washed with brine, dried over Na₂SO₄, concentrated to obtainthe crude product 26-6 (16.2 g) as a yellow oil. ESI-LCMS: m/z 801[M+H]⁺.

Preparation of Intermediate 26-7:

To a solution of the crude 26-6 (16.2 g) in pyridine (200 mL) was added2N NaOH (MeOH:H₂O=4:1) (50 mL) dropwise at 0° C. The mixture was stirredat 0° C. for 0.5 h and then neutralized with saturated NH₄Cl (aq) togive the pH=7˜8, and 500 mL H₂O. 800 mL of EA were added to separate thesolution, the aqueous was extracted with EA, the combined organic layerswere washed with brine, dried over Na₂SO₄, and concentrated to obtainthe crude product which was purified by MPLC to give 26-7 (9.3 g, 13.6mmol, 79.07% yield over two steps) as a white solid. ¹H-NMR (400 MHz,DMSO-d₆): δ=11.15 (s, 1H, exchanged with D₂O), 8.68 (s, 1H), 8.65 (s,1H), 8.07-8.05 (m, 2H), 7.67-7.63 (m, 1H), 7.57-7.53 (m, 2H), 7.44-7.41(m, 4H), 7.27 (d, J 9.2 Hz, 2H), 7.20-7.16 (m, 4H), 7.13-7.09 (m, 2H),6.72 (d, J 9.2 Hz, 2H), 6.08 (s, 1H), 5.93-5.83 (m, 1H), 5.27-5.22 (m,1H), 5.18 (t, J=4.0 Hz, 1H, exchanged with D₂O), 5.13-5.10 (m, 1H),4.11-3.99 (m, 4H), 3.64 (s, 3H), 3.46-3.42 (m, 1H), 3.38-3.32 (m, 1H),2.71 (d, J=10.8 Hz, 1H, exchanged with D₂O), 1.91 (d, J=4.4 Hz, 1H).ESI-LCMS: m/z 683 [M+H]⁺.

Preparation of 26-8:

Phosphitylation reagent (3.4 g, 11.4 mmol) was added under Ar to asolution of 26-7 (6.5 g, 9.5 mmol) and DCI (1.1 g, 9.5 mmol) in dry DCM(70 mL). The mixture was stirred at r.t. for 1 h. Then the reaction waswashed with 10% NaHCO₃(aq) and brine, dried over Na₂SO₄, andconcentrated to obtain the crude product which was purified byFlash-Prep-HPLC. This resulted in to give 26-8 (7.1 g, 8.0 mmol, 84.46%yield) as a white solid. ¹H-NMR (400 MHz, CDCl₃): δ=9.02 (br s, 1H),8.74 (d, J=10.4 Hz, 1H), 8.37 (d, J=108.4 Hz, 1H), 8.04 (t, J=7.2 Hz,2H), 7.65-7.61 (m, 1H), 7.57-7.53 (m, 2H), 7.51-7.48 (m, 4H), 7.41-7.38(m, 2H), 7.21-7.10 (m, 6H), 6.73-6.70 (m, 2H), 6.03 (d, J=2.4 Hz, 1H),5.91-5.79 (m, 1H), 5.31-5.21 (m, 2H), 5.17-5.13 (m, 1H), 4.40-4.25 (m,2H), 4.16-4.08 (m, 1H), 4.05-3.94 (m, 1H), 3.89-3.80 (m, 1H), 3.72 (dd,J=0.8, 10.8 Hz, 3H), 3.70-3.47 (m, 4H), 3.42-3.35 (m, 1H), 2.86 (dd,J=10.8, 23.2 Hz, 1H), 2.68-2.51 (m, 2H), 1.62 (dd, J=4.4, 210.8 Hz, 1H),1.26-1.18 (m, 12H). ³¹P NMR (162 MHz, CDCl₃): 148.85, 148.24. ESI-LCMS:m/z 883 [M+H]⁺.

Example 23

The synthesis of guanosine-based 2′-vinyl phosphoramidate 27-9 wasaccomplished as shown in scheme 27. The C3′-azide of compound 7-2 wasreduced to the corresponding amine to obtain 27-1. 3′-amine protectionwith N-Boc yielded intermediate 27-2. 2′-O-allylation was carried out byusing allylbromide in the presence of Ag₂O and NaI to form intermediate27-3. Boc protection at the 3′-NH position was found to afford higheralkylation yields as compared to MMTr for this particular case. MMTrcleavage of the exocyclic amino to give 27-4 followed by deprotectionwith iBuCl gave compound 27-5. Then, deprotection of the 3′amine gave27-6 which after reprotection with MMTrCl yielded 27-7. Cleavage of the5′-tol group to form 27-8 and final phosphitylation gave target compound27-9.

Preparation of Intermediate (27-1):

To a solution of 7-2 (50.5 g, 69.7 mmol) in THF (500 mL) were added PPh₃(27.4 g, 104.5 mmol) and H₂O (1.3 g, 69.7 mmol). The mixture was stirredat 60° C. for 15 h. The reaction was concentrated and the residue waspurified on silica gel to give 27-1 (41.1 g, 59.3 mmol, 85.08% yield) asa white solid. ESI-LCMS: m/z 691 [M+H]⁺.

Preparation of Intermediate (27-2):

To a solution of 27-1 (41.1 g, 59.3 mmol) in a mixture of THF (300 mL)and sat. NaHCO₃(aq) (200 mL) was added Boc₂O (15.5 g, 71.2 mmol). Themixture was stirred at room temperature for 2 h, extracted with EA,washed with brine and dried over anhydrous Na₂SO₄. Finally, it wasconcentrated to obtain the crude product which was purified on silicagel to give 27-2 (46.2 g, 58.2 mmol, 98.00% yield) as a white solid.ESI-LCMS: m/z 791 [M+H]⁺.

Preparation of Intermediate (27-3):

Vinyl bromide (45 g, 56.8 mmol) and 27-2 (10.3 g, 85.3 mmol) in dry DMF(400 mL), were added Ag₂O (19.7 g, 85.3 mmol) and NaI (12.8 g, 85.3mmol), the mixture was stirred at room temperature for 0.5 h, pouredinto ice water, extracted with EA, washed with brine and dried overanhydrous Na₂SO₄, concentrated to obtain the crude product which waspurified on silica gel to give 27-3 (35.3 g, 42.4 mmol, 74.66% yield) asa white solid. ESI-LCMS: m/z 831 [M+H]⁺.

Preparation of Intermediate (27-4):

TCA (100 g, 42.1 mmol) was added to solution of 27-3 (35 g, 42.1 mmol)in DCM (400 mL) and the mixture was stirred at room temperature for 4 h.

After this time, the reaction mixture was neutralized with saturatedNaHCO₃(aq) to give pH=8-9.

EA was added and washed with brine. The organic layers were dried overNa₂SO₄ and concentrated to obtain the crude product which was purifiedon silica gel to give 27-4 (18.5 g, 33.1 mmol, 78.61% yield) as a whitesolid. ESI-LCMS: m/z 559 [M+Na]⁺.

Preparation of Intermediate (27-5):

iBuCl (5.2 g, 48.3 mmol) was added dropwise to a solution of 27-4 (18 g,32.2 mmol) in pyridine (200 mL) at 0° C. After the mixture was stirredat 0° C. for 0.5 h, 500 mL H₂O and 800 mL EA were added to separate thesolution. The aqueous phase was extracted with EA, the combined organiclayer was washed with brine, dried over Na₂SO₄ and finally concentratedto obtain the crude product which was purified on silica gel to give27-5 (18.1 g, 28.77 mmol, 89.35% yield) as a white solid. ESI-LCMS: m/z629 [M+H]⁺.

Preparation of Intermediate (27-6):

27-5 (18 g, 28.6 mmol) was dissolved in a mixture of TFA (180 mL) andH₂O (60 mL). The reaction was stirred at room temperature for 15 h andneutralized with saturated NaHCO₃(aq). The crude was extracted with EA,washed with brine, and dried over anhydrous Na₂SO₄. After concentration,purification on silica gel afforded 27-6 (12.5 g, 24.5 mmol, 85.57%yield) as a white solid. ESI-LCMS: m/z 511 [M+H]⁺.

Preparation of Intermediate (27-7):

To a solution of 27-6 (12.5 g, 24.5 mmol) in anhydrous DCM (150 mL),were added collidine (5.9 g, 49.0 mmol), AgNO₃ (6.2 g, 36.7 mmol), andMMTr-Cl (11.3 g, 36.7 mmol) and the mixture was stirred at r.t. for 1 hunder N₂. After the reaction reached completion, the crude reactionmixture was filtered, washed with H₂O, dried over Na₂SO₄, andconcentrated to give a residue which was purified on silica gel toafford the crude product 27-7 (18.5 g, 23.6 mmol, 96.56% yield) as awhite solid. ESI-LCMS: m/z 783 [M+H]⁺.

Preparation of Intermediate (27-8):

To a solution of crude 27-7 (18.5 g, 23.6 mmol) in pyridine (200 mL) 2NNaOH was added dropwise (in MeOH:H₂O=4:1) (50 mL) dropwise at 0° C., themixture was stirred at 0° C. for 15 min. The mixture was neutralizedwith saturated NH₄Cl (aq) to give pH=7˜8, and 500 mL H₂O and 800 mL EAwere added. The aqueous phase was extracted by EA, the combined organiclayers were washed with brine, dried over Na₂SO₄, and concentrated toobtain the crude product which was purified by MPLC to give 27-8 (12.1g, 18.2 mmol, 77.21%) as a white solid. ¹H-NMR (400 MHz, CDCl₃): δ=12.13(s, 1H, exchanged with D₂O), 11.34 (s, 1H, exchanged with D₂O), 8.11 (s,1H), 7.47-7.43 (m, 4H), 7.28-7.21 (m, 6H), 7.17-7.13 (m, 2H), 6.77-6.75(m, 2H), 5.89-5.80 (m, 2H), 5.25-5.20 (m, 1H), 5.13-5.09 (m, 1H),4.03-3.92 (m, 4H), 3.65 (s, 3H), 3.46-3.41 (m, 1H), 3.31-3.24 (m, 1H),2.87-2.80 (m, 1H), 2.73 (d, J=10.0 Hz, 1H, exchanged with D₂O), 1.91 (d,J=4.8 Hz, 1H), 1.16 (dd, J=6.8, 11.2 Hz, 6H). ESI-LCMS: m/z 665 [M+H]⁺.

Preparation of (27-9):

To a solution of 27-8 (7.5 g, 11.3 mmol) and DCI (1.5 g, 12.4 mmol) indry DCM (80 mL) was added the phosphitylation reagent (4.4 g, 14.7 mmol)under Ar. The mixture was stirred at r.t. for 1 h, washed with 10%NaHCO₃(aq) and brine, dried over Na₂SO₄ and concentrated to obtain thecrude product which was purified by Flash-Prep-HPLC. This resulted in togive 27-9 (7.4 g, 8.5 mmol, 75.79% yield) as a white solid. ¹H-NMR (400MHz, CDCl₃): δ=7.90 (d, J=119.2 Hz, 1H), 754-7.51 (m, 4H), 7.42-7.37 (m,2H), 7.28-7.12 (m, 6H), 6.78-6.72 (m, 2H), 5.79-5.65 (m, 2H), 5.18-5.04(m, 2H), 4.30-4.09 (m, 3H), 3.93-3.33 (m, 10H), 2.86 (dd, J=10.0, 28.8Hz, 1H), 2.72-2.52 (m, 3H), 2.10 (dd, J=4.8, 211.6 Hz, 1H), 1.28-1.11(m, 18H). ³¹P NMR (162 MHz, CDCl₃): 149.14, 148.15. ESI-LCMS: m/z 865[M+H]⁺.

Example 24

The synthesis of the 2′-O-allyl uridine phosphoramidite 28-5 wasachieved as shown below in scheme 28. Briefly, MMTrCl-mediatedprotection of the 3′NH₂ group of 8-5 afforded compound 28-4 that wasalkylated with allylbromide to give 28-2. Deprotection of the propiolategroup was accomplished with pyrrolidine leading to the formation of28-3. Then, cleavage of the 5′-ester gave intermediate 28-4 that wasphosphitylated to afford target phosphoramidite 28-5.

Preparation of Intermediate (28-1):

MMTrCl (15.3 g, 49.6 mmol) was added under Ar to a solution of 8-5 (19.0g, 41.3 mmol) in pyridine (300 mL), the mixture was stirred at roomtemperature for 1 h. Quenched with MeOH and concentrated to obtain aresidue which was purified on silica gel to give 7 (24.5 g, 33.4 mmol,80.97% yield) as a white solid. ¹H-NMR (400 MHz, DMSO-d₆): δ=8.08 (d,J=14.8 Hz, 1H), 7.59-7.48 (m, 7H), 7.35-7.28 (m, 4H), 7.23-7.16 (m, 4H),7.12-7.03 (m, 2H), 6.87 (d, J=14.8 Hz, 1H), 6.73 (d, J=4.8 Hz, 1H), 5.93(d, J=4.4 Hz, 1H, exchanged with D₂O), 5.42-5.40 (m, 2H), 4.73 (d,J=12.4 Hz, 1H), 4.61 (dd, J=2.8, 12.8 Hz, 1H), 4.28-4.19 (m, 3H), 3.58(s, 3H), 3.13-3.04 (m, 2H), 2.38 (s, 3H), 2.15 (t, J=4.0 Hz, 1H), 1.27(t, J=7.2 Hz, 3H). ESI-LCMS: m/z 732 [M+H]⁺.

Preparation of Intermediate (28-2):

To a solution of 28-1 (22.5 g, 30.7 mmol) and allyl bromide (7.4 g, 61.5mmol) in dry DMF (250 mL), were added NaI (9.2 g, 61.5 mmol) and Ag₂O(10.7 g, 46.1 mmol) under Ar, the mixture was stirred at roomtemperature for 0.5 h. The reaction was poured into ice water, extractedwith EA, washed with brine and dried over anhydrous Na₂SO₄, concentratedto obtain the crude product which was purified on silica gel to give28-2 (18.8 g, 24.3 mmol, 79.22% yield) as a white solid. ¹H-NMR (400MHz, DMSO-d₆): δ=8.06 (d, J=14.8 Hz, 1H), 7.59 (t, J=8.0 Hz, 3H), 7.50(t, J=8.4 Hz, 4H), 7.33 (d, J=8.4 Hz, 2H), 7.27 (d, J=8.0 Hz, 2H),7.22-7.15 (m, 4H), 7.10 (t, J=7.2 Hz, 1H), 7.04 (t, J=7.6 Hz, 1H), 6.87(dd, J=2.4, 22.8 Hz, 1H), 6.72 (d, J=8.8 Hz, 2H), 5.88-5.78 (m, 1H),5.38 (d, J=8.0 Hz, 1H), 5.22 (dd, J=1.6, 17.2 Hz, 1H), 5.11-5.08 (m,1H), 4.87-4.75 (m, 2H), 4.34 (d, J=10.4 Hz, 1H), 4.20 (dd, J=7.2, 14.4Hz, 2H), 4.06-3.99 (m, 1H), 3.55 (s, 3H), 3.32-3.30 (m, 2H), 2.79 (d,J=10.4 Hz, 1H, exchanged with D₂O), 2.37 (s, 3H), 1.84 (d, J=4.4 Hz,1H), 1.26 (t, J=7.2 Hz, 3H). ESI-LCMS: m/z 772 [M+H]⁺.

Preparation of Intermediate (28-3):

Pyrrolidine (1.8 g, 25.9 mmol) was added to a solution of 28-2 (20.0 g,25.9 mmol) in acetonitrile (200 mL). The mixture was stirred at roomtemperature for 15 h. The solvent was evaporated and the residue waspurified on silica gel to give 28-3 (15.5 g, 23.0 mmol, 88.81% yield) asa slightly yellow solid. ESI-LCMS: m/z 696 [M+Na]⁺.

Preparation of Intermediate (28-4):

A mixture of 28-3 (6.0 g, 8.9 mmol) and 2 N NaOH (in MeOH: H₂O=4:1) (60mL) was stirred at r.t. for 0.5 h. After TLC showed that 28-3 wasconsumed completely, 28-4 was extracted with DCM and washed with brine,dried over Na₂SO₄, and concentrated to obtain the crude product whichwas purified by MPLC to give 28-4 (4.3 g, 7.7 mmol, 87.05%) as a whitesolid. ¹H-NMR (400 MHz, DMSO-d₆): δ=11.27 (s, 1H, exchanged with D₂O),7.97 (d, J=8.0 Hz, 1H), 7.47-7.44 (m, 4H), 7.34 (d, J=8.8 Hz, 2H), 7.27(t, J=7.2 Hz, 4H), 7.21-7.17 (m, 2H), 6.84 (d, J=8.8 Hz, 2H), 5.86-5.76(m, 1H), 5.25-5.49 (m, 2H), 5.20-5.15 (m, 1H), 5.08-5.05 (m, 1H),4.06-4.01 (m, 2H), 3.96-3.90 (m, 2H), 3.72 (s, 3H), 3.30-3.25 (m, 1H),3.16-3.09 (m, 1H), 2.69 (d, J=10.4 Hz, 1H, exchanged with D₂O), 1.57 (d,J=4.4 Hz, 1H). ESI-LCMS: m/z 556 [M+H]⁺.

Preparation of (28-5):

To a solution of 28-4 (4.3 g, 7.7 mmol) and DCI (999 mg, 8.5 mmol) indry DCM (40 mL) was added 28-4 (3.0 g, 10.0 mmol) under Ar. The mixturewas stirred at r.t. for 1 h. The reaction was washed with 10%NaHCO₃(aq.) and brine, dried over Na₂SO₄ and concentrated to obtain thecrude product which was purified Flash-Prep-HPLC. 28-5 (4.4 g, 5.8 mmol,75.68% yield) was obtained as a white solid. ¹H-NMR (400 MHz, CDCl₃):δ=7.80 (dd, J=8.0, 182.4 Hz, 1H), 7.55-7-43 (m, 6H), 7.30-7.18 (m, 6H),6.82-6.78 (m, 2H), 5.84-5.74 (m, 1H), 5.66 (d, J=4.8 Hz, 1H), 5.60 (d,J=8.0 Hz, 1H), 5.22-5.17 (m, 1H), 5.11-5.08 (m, 1H), 4.32-4.29 (m, 1H),4.19-4.01 (m, 3H), 3.89-3.81 (m, 1H), 3.78 (s, 3H), 3.66-3.46 (m, 3H),3.39-3.35 (m, 1H), 3.22-3.01 (m, 1H), 2.94-2.88 (m, 1H), 2.64-2.44 (m,1H), 1.52 (dd, J=4.8, 140.0 Hz, 1H), 1.25-1.15 (m, 12H). ³¹P-NMR (162MHz, CDCl₃): 149.16, 148.20. ESI-LCMS: m/z 756 [M+H]⁺.

Example 25

Preparation of Intermediate (29-1):

DMAP (3.6 g, 29.7 mmol), TEA (3.0 g, 29.6 mmol, 4 mL) and TPSCl (6.7 g,22.2 were added under Ar to solution of 28-3 (10.0 g, 14.8 mmol) in dryacetonitrile (100 mL) were added mmol). After the mixture was stirred atr.t. for 1 h, conc. NH₃.H₂O (25 mL) was added, and the reaction wasstirred at r.t. for another 15 h. Upon of completion, the solvent wasremoved, and the residue was dissolved in EA, washed with sat. NH₄Cl(aq) and brine, dried over anhydrous Na₂SO₄, and concentrated to obtainthe crude product which was purified on silica gel to give 29-1 (8.9 g,13.2 mmol, 89.14% yield) as a pale yellow solid. ¹H-NMR (400 MHz,DMSO-d₆): δ=7.59 (d, J=7.6 Hz, 2H, exchanged with D₂O), 7.50-7.43 (m,5H), 7.35-7.29 (m, 4H), 7.22-7.17 (m, 6H), 7.14-7.06 (m, 2H), 6.71 (d,J=9.2 Hz, 2H), 5.88-5.79 (m, 1H), 5.55 (s, 1H), 5.39 (d, J=7.2 Hz, 1H),5.22 (dd, J=1.6, 17.2 Hz, 1H), 5.08 (dd, J=1.2, 10.4 Hz, 1H), 4.88-4.76(m, 2H), 4.31-4.28 (m, 1H), 4.09-4.04 (m, 1H), 3.58 (s, 3H), 3.29-3.25(m, 1H), 3.20-3.14 (m, 1H), 2.79 (d, J=9.2 Hz, 1H, changed with D₂O),2.41 (s, 3H), 1.61 (d, J=4.4 Hz, 1H). ESI-LCMS: m/z 673 [M+H]⁺.

Preparation of Intermediate (29-2):

To a solution of 29-1 (8.5 g, 12.6 mmol) in pyridine (80 mL), BzCl wasadded dropwise at 0° C. (2.1 g, 15.1 mmol). After the mixture wasstirred for 1 h at r.t., 300 mL H₂O and 500 mL EA were added. Theaqueous layer was extracted by EA, the combined organic layer was washedwith brine, dried over Na₂SO₄, and concentrated to obtain the crudeproduct 29-2 (11.30 g) as a yellow oil. ESI-LCMS: m/z 777 [M+H]⁺.

Preparation of Intermediate (29-3):

2N NaOH (60 mL, MeOH:H₂O=4:1) was added dropwise at 0° C. to a solutionof the crude product 29-2 (12.0 g, 15.4 mmol) in pyridine (120 mL). Themixture was stirred at 0° C. for 15 min and then neutralized withsaturated NH₄Cl (aq.). 500 mL H₂O and 800 mL EA were added. The aqueousphase was extracted by EA, the combined organic layers were washed withbrine, dried over Na₂SO₄, and concentrated to obtain the crude productwhich was purified on silica gel to give 29-3 (6.5 g, 9.8 mmol, 66.2%yield over two steps) as a white solid. ¹H-NMR (400 MHz, CDCl₃): δ=8.47(d, J=7.6 Hz, 1H), 7.92 (d, J=7.6 Hz, 1H), 7.65-7.60 (m, 1H), 7.54-7.50(m, 6H), 7.47-7.44 (m, 2H), 7.29-7.22 (m, 6H), 6.85-6.78 (m, 2H),5.90-5.80 (m, 1H), 5.74 (s, 1H), 5.28-5.23 (m, 1H), 5.14-5.11 (m, 1H),4.30-4.25 (m, 3H), 4.18-4.15 (m, 1H), 3.79 (s, 3H), 3.54-3.48 (m, 1H),3.13-3.07 (m, 1H), 2.84 (d, J=10.8 Hz, 1H), 1.92 (d, J=4.4 Hz, 1H).ESI-LCMS: m/z 659 [M+H]⁺.

Preparation of (29-4):

CEP[N(iPr)₂]₂ (2.4 g, 8.1 mmol) was added under Ar to a solution of 29-3(3.8 g, 5.8 mmol) and DCI (816 mg, 6.9 mmol) in dry DCM (40 mL). Themixture was stirred at r.t. for 1 h. The reaction mixture was washedwith 10% NaHCO₃(aq.) and brine, dried over Na₂SO₄ and concentrated toobtain the crude product which was purified Flash-Prep-HPLC. 29-4 (3.9g, 4.5 mmol, 78.69% yield) was obtained as a white solid. ¹H-NMR (400MHz, CDCl₃): δ=8.67 (d, J=7.6 Hz, 1H), 7.94-7-91 (m, 2H), 7.64 (t, J=7.2Hz, 1H), 7.56-7.42 (m, 8H), 7.28-7.20 (m, 6H), 6.81-6.77 (m, 2H),5.87-5.78 (m, 1H), 5.74 (d, J=4.0 Hz, 1H), 5.25-5.20 (m, 1H), 5.11-5.08(m, 1H), 4.43-4.34 (m, 1H), 4.27-4.21 (m, 2H), 4.15-4.09 (m, 1H),3.94-3.84 (m, 1H), 3.79 (d, J=1.6 Hz, 3H), 3.73-3.56 (m, 3H), 3.46-3.42(m, 1H), 3.22-3.03 (m, 1H), 2.87 (t, J=10.0 Hz, 1H), 2.69 (t, J=6.4 Hz,1H), 2.63-2.54 (m, 1H), 1.72 (dd, J=4.4, 105.6 Hz, 1H), 1.30-1.22 (m,12H). ³¹P-NMR (162 MHz, CDCl₃): 149.00, 148.08. ESI-LCMS: m/z 859[M+H]⁺.

Example 26—Synthesis of Oligonucleotides

The modified oligonucleotides are synthesized on an ABI-394 synthesizerusing the 93-step cycle written with modifications to deblock, couplingand wait steps. The solid support is 3′-NHTr-5′-LCAA-CPG. Eacholigonucleotide is individually synthesized using methods describedherein.

The modified oligonucleotides can be purified by anion-exchange HPLC.The buffers are, e.g., 20 mM sodium phosphate in 10% CH₃CN, pH 8.5(buffer A) and 20 mM sodium phosphate in 10% CH₃CN, 1.8 M NaBr, pH 8.5(buffer B). Fractions containing full-length oligonucleotides arepooled, desalted, and lyophilized.

The purified dry oligomer are then desalted using Sephadex G-25 M(Amersham Biosciences). The cartridge is conditioned with 10 mL ofdeionized water thrice. Finally the purified oligomer is dissolvedthoroughly in RNAse free water is applied to the cartridge with veryslow drop wise elution. The salt free oligomer is eluted with deionizedwater directly into a screw cap vial.

Approximately 0.10 OD of oligomer is dissolved in water and thenpipetted in special vials for IEX-HPLC and LC/MS analysis. AnalyticalHPLC and ES LC-MS establishes the integrity of the oligonucleotides. Thepurity and molecular weight are determined by HPLC analysis (60° C.,IEX-Thermo DNAPac PA-100, A-25 mM sodium phosphate 10% acetonitrile pH11, B-1.8 M NaBr 25 mM sodium phosphate 10% acetonitrile pH 11;RPIP-Waters XBridge OST C18, A-100 mM HFIP 7 mM TEA B-7:3methanol/acetonitrile) and ESI-MS analysis using Promass Deconvolutionfor Xcalibur (Novatia, Newtown, Pa.).

1. A compound represented by Formula (I):

wherein X is O or NH; Y is selected from the group consisting of O-PG,—O(CR⁴ ₂)_(a)CR⁴ ₃, —O(CR⁴ ₂)_(b)OCR⁴ ₃ and —O(CR⁴ ₂)_(b)—CR⁴═CR⁴ ₂; Zis H; PG is a protecting group; B is a natural or an unmodifiednucleobase or a modified nucleobase or a protected version thereof; eachR¹ is independently C₁₋₆ alkyl or cycloalkyl; R² is CH₂CH₂CN or C₁₋₆alkyl; or one R¹ and R² together form an optionally substituted C₁₋₆cycloalkyl; R³ is H or PG; R⁴ is independently in each instance H or F;a is an integer of 0-2; and b is an integer of 1-3, wherein when X is O,then Y is —O(CR⁴ ₂)_(a)CR⁴ ₃, —O(CR⁴ ₂)_(b)OCR⁴ ₃ or —O(CR⁴₂)_(b)—CR⁴═CR⁴ ₂.
 2. The compound of claim 1, wherein X is NH and Y isO-PG, OEt or O-methoxyethoxy.
 3. The compound of claim 1, wherein R′ isC₂₋₃ alkyl.
 4. The compound of claim 1, wherein R′ is isopropyl.
 5. Thecompound of claim 1, wherein R³ is a trityl protecting group.
 6. Thecompound of claim 1, wherein R³ is monomethoxytrityl (MMTr) or tritolylwhen X is NH and 4,4′-dimethoxytrityl (DMTr) or tritolyl when X is O. 7.The compound of claim 1, wherein B is selected from adenine (A), guanine(G), thymine (T), cytosine (C), uracil (U) and 5-methylcytosine(5-me-C), or a protected version thereof.
 8. The compound of claim 1,wherein B is selected from 6-N-benzoyladenosine (A^(Bz)),4-N-Benzoylcytidine (C^(Bz)), and 2-N-isobutyrylguanosine (G^(iBu)). 9.A composition comprising the compound of claim 1 and one or morereactants or solvents or impurities.
 10. (canceled)
 11. A method ofmaking an oligonucleotide comprising a modified nucleoside comprisingreacting the compound of claim 1 to form the oligonucleotide.
 12. Acompound represented by Formula (II):

wherein X is O or NH; Z is H; B is a natural or an unmodified nucleobaseor a modified nucleobase or a protected version thereof; R¹ isindependently a CI-6 alkyl or cycloalkyl; R² is CH₂CH₂CN or a C₁₋₆alkyl; or one R¹ and R² together form an optionally substituted C₁₋₆cycloalkyl; R³ is H or a protecting group; A is —(CR′R′)₁₋₂—; and R′ isindependently in each instance H or Me.
 13. The compound of claim 12,wherein X is NH and Z is H.
 14. The compound of claim 12, wherein R¹ isa C₂₋₃ alkyl.
 15. The compound of claim 12, wherein R′ is isopropyl. 16.The compound of claim 12, wherein R³ is a trityl protecting group. 17.The compound of claim 12, wherein R³ is monomethoxytrityl (MMTr) ortritolyl when X is NH and 4,4′-dimethoxytrityl (DMTr) or tritolyl when Xis O.
 18. The compound of claim 12, wherein B is selected from adenine(A), guanine (G), thymine (T), cytosine (C), uracil (U) and5-methylcytosine (5-me-C), or a protected version thereof.
 19. Thecompound of claim 12, wherein B is selected from 6-N-benzoyladenosine(A^(Bz)), 4-N-Benzoylcytidine (C^(Bz)), and 2-N-isobutyrylguanosine(G^(iBu)).
 20. A composition comprising the compound of claim 12 and oneor more reactants or solvents or impurities.
 21. (canceled)
 22. A methodof making an oligonucleotide comprising a modified nucleoside, saidmethod comprising reacting the compound of claim 12 to form theoligonucleotide. 23-24. (canceled)